Compositions and methods for the treatment and diagnosis of cardiovascular disease

ABSTRACT

The present invention relates to methods and compositions for the treatment and diagnosis of cardiovascular disease, including, but not limited to, atherosclerosis, ischemia/reperfusion, hypertension, restenosis, and arterial inflammation. Specifically, the present invention identifies and describes genes which are differentially expressed in cardiovascular disease states, relative to their expression in normal, or non-cardiovascular disease states, and/or in response to manipulations relevant to cardiovascular disease. Further, the present invention identifies and describes genes via the ability of their gene products to interact with gene products involved in cardiovascular disease. Still further, the present invention provides methods for the identification and therapeutic use of compounds as treatments of cardiovascular disease moreover, the present invention provides methods for the diagnostic monitoring of patients undergoing clinical evaluation for the treatment of cardiovascular disease, and for monitoring the efficacy of compounds in clinical trials. Additionally, the present invention describes methods for the diagnostic evaluation and prognosis of various cardiovascular diseases, and for the identification of subjects exhibiting a predisposition to such conditions.

This is a division of application Ser. No. 08/799,910, filed Feb. 13,1997, which claims benefit of priority under 35 U.S.C. §119(e) ofprovisional application Ser. No. 60/011,787, filed Feb. 16, 1996, eachof which is hereby incorporated by reference in its entirety.

1. INTRODUCTION

2. BACKGROUND OF THE INVENTION

3. SUMMARY OF THE INVENTION

4. DESCRIPTION OF THE FIGURES

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES

5.1.1. PARADIGMS FOR THE IDENTIFICATION OF DIFFERENTIALLY EXPRESSEDGENES

5.1.1.1. FOAM CELL PARADIGM--1

5.1.1.2. FOAM CELL PARADIGM--2

5.1.1.3. FOAM CELL PARADIGM--3

5.1.1.4. IN VIVO MONOCYTE PARADIGM

5.1.1.5. ENDOTHELIAL CELL--IL-1 PARADIGM

5.1.1.6. ENDOTHELIAL CELL--SHEAR STRESS PARADIGM

5.1.2. ANALYSIS OF PARADIGM MATERIAL

5.2. IDENTIFICATION OF PATHWAY GENES

5.3. CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED AND PATHWAY GENES

5.4. DIFFERENTIALLY EXPRESSED AND PATHWAY GENES

5.4.1. DIFFERENTIALLY EXPRESSED AND PATHWAY GENE SEQUENCES

5.4.2. DIFFERENTIALLY EXPRESSED AND PATHWAY GENE PRODUCTS

5.4.3.DIFFERENTIALLY EXPRESSED OR PATHWAY GENE PRODUCT ANTIBODIES

5.4.4. CELL- AND ANIMAL-BASED MODEL SYSTEMS

5.4.4.1. ANIMAL-BASED SYSTEMS

5.4.4.2. CELL-BASED ASSAYS

5.5. SCREENING ASSAYS FOR COMPOUNDS THAT INTERACT WITH THE TARGET GENEPRODUCT AND/OR MODULATE TARGET GENE EXPRESSION

5.5.1. IN VITRO SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO THE TARGETGENE PRODUCT

5.5.2. ASSAYS FOR CELLULAR OR EXTRACELLULAR PROTEINS THAT INTERACT WITHTHE TARGET GENE PRODUCT

5.5.3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH INTERACTION BETWEENTARGET GENE PRODUCT AND OTHER COMPOUNDS

5.5.4. ASSAYS FOR AMELIORATION OF CARDIOVASCULAR DISEASE SYMPTOMS

5.5.5. MONITORING OF EFFECTS DURING CLINICAL TRIALS

5.5.6. ASSAYS FOR COMPOUNDS THAT MODULATE EXPRESSION OF TARGET GENES

5.6 COMPOUNDS AND METHODS FOR TREATMENT OF CARDIOVASCULAR DISEASE

5.6.1. COMPOUNDS THAT INHIBIT EXPRESSION, SYNTHESIS OR ACTIVITY OFMUTANT TARGET GENE ACTIVITY

5.6.1.1. INHIBITORY ANTISENSE, RIBOZYME, TRIPLE HELIX, AND GENEINACTIVATION APPROACHES

5.6.1.2. ANTIBODIES FOR TARGET GENE PRODUCTS

5.6.2. METHODS FOR RESTORING OR ENHANCING TARGET GENE ACTIVITY

5.7 PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION

5.7.1. EFFECTIVE DOSE

5.7.2. FORMULATIONS AND USE

5.8 DIAGNOSIS OF CARDIOVASCULAR DISEASE ABNORMALITIES

5.8.1. DETECTION OF FINGERPRINT GENE NUCLEIC ACIDS

5.8.2. DETECTION OF FINGERPRINT GENE PEPTIDES

5.8.3. IMAGING CARDIOVASCULAR DISEASE CONDITIONS

6. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN RESPONSETO PARADIGM A: IN VITRO FOAM CELL PARADIGM

6.1 MATERIALS AND METHODS

6.1.1. CELL ISOLATION AND CULTURING

6.1.2. ANALYSIS OF PARADIGM MATERIAL

6.1.3. CHROMOSOMAL LOCALIZATION OF TARGET GENES

6.2. RESULTS

7. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN RESPONSETO PARADIGM D: ENDOTHELIAL CELL SHEAR STRESS

7.1. MATERIALS AND METHODS

7.2. RESULTS

8. EXAMPLE: USE OF GENES UNDER PARADIGM A AS SURROGATE MARKERS INCLINICAL TRIALS

8.1 TREATMENT OF PATIENTS AND CELL ISOLATION

8.2 ANALYSIS OF SAMPLES

9. EXAMPLE: IMAGING OF A CARDIOVASCULAR DISEASE CONDITION

9.1. MONOCLONAL CONJUGATED ANTIBODIES

9.2 ADMINISTRATION AND DETECTION OF IMAGING AGENTS

10. POLYCLONAL ANTIBODIES TO TARGET GENE PEPTIDE SEQUENCES

11. EXAMPLE: THE RCHD534 AND FCHD540 GENE PRODUCTS

11.1. MATERIALS AND METHODS

11.1.1. YEAST STRAINS, MEDIA, AND MICROBIOLOGICAL TECHNIQUES

11.1.2. PLASMID AND YEAST STRAIN CONSTRUCTION

11.1.3. TWO-HYBRID SCREENING

11.1.4. PAPER FILTER BETA-GALACTOSIDASE ASSAYS

11.2. RESULTS

11.2.1. STRONG PHYSICAL INTERACTION OF RCHD534 AND FCHD540 MEASURED BYTWO-HYBRID ASSAY

11.2.2. IDENTIFICATION OF PROTEINS THAT PHYSICALLY INTERACT WITH FCHD540

11.2.3. RETRANSFORMATION AND SPECIFICITY TESTING OF TCHV03A AND TCHVR4A

11.3. FURTHER ANALYSIS OF RCHD534 AND FCHD540 FUNCTION

11.3.1. TISSUE EXPRESSION PATTERNS

11.3.2. CELLULAR LOCALIZATION

11.3.3. PROTEIN INTERACTIONS IN HUMAN CELLS

11.3.4. EFFECT OF EXPRESSION ON TGF-B SIGNALLING

12. ANTISENSE AND RIBOZYME MOLECULES FOR INHIBITION OF RCHD534 ANDFCHD540 EXPRESSION

13. DEPOSIT OF MICROORGANISMS

1. INTRODUCTION

The present invention relates to methods and compositions for thetreatment and diagnosis of cardiovascular disease, including, but notlimited to, atherosclerosis, ischemia/reperfusion, hypertension,restenosis, and arterial inflammation Genes which are differentiallyexpressed in cardiovascular disease states, relative to their expressionin normal, or non-cardiovascular disease states are identified Genes arealso identified via the ability of their gene products to interact withother gene products involved in cardiovascular disease The genesidentified may be used diagnostically or as targets for therapeuticintervention. In this regard, the present invention provides methods forthe identification and therapeutic use of compounds in the treatment anddiagnosis of cardiovascular disease Additionally, methods are providedfor the diagnostic monitoring of patients undergoing clinical evaluationfor the treatment of cardiovascular disease, for monitoring the efficacyof compounds in clinical trials, and for identifying subjects who may bepredisposed to cardiovascular disease

2. BACKGROUND OF THE INVENTION

Cardiovascular disease is a major health risk throughout theindustrialized world. Atherosclerosis, the most prevalent ofcardiovascular diseases, is the principal cause of heart attack, stroke,and gangrene of the extremities, and thereby the principal cause ofdeath in the United States. Atherosclerosis is a complex diseaseinvolving many cell types and molecular factors (for a detailed review,see Ross, 1993, Nature 362: 801-809). The process, in normalcircumstances a protective response to insults to the endothelium andsmooth muscle cells (SMCs) of the wall of the artery, consists of theformation of fibrofatty and fibrous lesions or plaques, preceded andaccompanied by inflammation The advanced lesions of atherosclerosis mayocclude the artery concerned, and result. from an excessiveinflammatory-fibroproliferative response to numerous different forms ofinsult. For example, shear stresses are thought to be responsible forthe frequent occurrence of atherosclerotic plaques in regions of thecirculatory system where turbulent blood flow occurs, such as branchpoints and irregular structures.

The first observable event in the formation of an atherosclerotic plaqueoccurs when blood-borne monocytes adhere to the vascular endotheliallayer and transmigrate through to the sub-endothelial space. Adjacentendothelial cells at the same time produce oxidized low densitylipoprotein (LDL). These oxidized LDL's are then taken up in largeamounts by the monocytes through scavenger receptors expressed on theirsurfaces. In contrast to the regulated pathway by which native LDL(nLDL) is taken up by nLDL specific receptors, the scavenger pathway ofuptake is not regulated by the monocytes.

These lipid-filled monocytes are called foam cells, and are the majorconstituent of the fatty streak Interactions between foam cells and theendothelial and SMCs which surround them lead to a state of chroniclocal inflammation which can eventually lead to smooth muscle cellproliferation and migration, and the formation of a fibrous plaque. Suchplaques occlude the blood vessel concerned and thus restrict the flow ofblood, resulting in ischemia.

Ischemia is a condition characterized by a lack of oxygen supply intissues of organs due to inadequate perfusion. Such inadequate perfusioncan have number of natural causes, including atherosclerotic orrestenotic lesions, anemia, or stroke, to name a few. Many medicalinterventions, such as the interruption of the flow of blood duringbypass surgery, for example, also lead to ischemia. In addition tosometimes being caused by diseased cardiovascular tissue, ischemia maysometimes affect cardiovascular tissue, such as in ischemic heartdisease. Ischemia may occur in any organ, however, that is suffering alack of oxygen supply.

The most common cause of ischemia in the heart is atheroscleroticdisease of epicardial coronary arteries. By reducing the lumen of thesevessels, atherosclerosis causes an absolute decrease in myocardialperfusion in the basal state or limits appropriate increases inperfusion when the demand for flow is augmented. Coronary blood flow canalso be limited by arterial thrombi, spasm, and, rarely, coronaryemboli, as well as by ostial narrowing due to luetic aortitis.Congenital abnormalities, such as anomalous origin of the left anteriordescending coronary artery from the pulmonary artery, may causemyocardial ischemia and infarction in infancy, but this cause is veryrare in adults. Myocardial ischemia can also occur if myocardial oxygendemands are abnormally increased, as in severe ventricular hypertrophydue to hypertension or aortic stenosis. The latter can be present withangina that is indistinguishable from that caused by coronaryatherosclerosis A reduction in the oxygen-carrying capacity of theblood, as in extremely severe anemia or in the presence ofcarboxy-hemoglobin, is a rare cause of myocardial ischemia. Notinfrequently, two or more causes of ischemia will coexist, such as anincrease in oxygen demand due to left ventricular hypertrophy and areduction in oxygen supply secondary to coronary atherosclerosis.

The principal surgical approaches to the treatment of ischemicatherosclerosis are bypass grafting, endarterectomy, and percutaneoustranslumenal angioplasty (PCTA). The failure rate after these approachesdue to restenosis, in which the occlusions recur and often become evenworse, is extraordinarily high (30-50%). It appears that much of therestenosis is due to further inflammation, smooth muscle accumulation,and thrombosis

A modified balloon angioplasty approach was used to treat arterialrestenosis in pigs by gene therapy (Ohno et al., 1994, Science 265:781-784). A specialized catheter was used to introduce a recombinantadenovirus carrying the gene encoding thymidine kinase (tk) into thecells at the site of arterial blockage. Subsequently, the pigs weretreated with ganciclovir, a nucleoside analog which is converted by tkinto a toxic form which kills cells when incorporated into DNA. Treatedanimals had a 50% to 90% reduction in arterial wall thickening withoutany observed local or systemic toxicities.

Because of the presumed role of the excessiveinflammatory-fibroproliferative response in atherosclerosis andischemia, a number of researchers have investigated, in the context ofarterial injury, the expression of certain factors involved ininflammation, cell recruitment and proliferation. These factors includegrowth factors, cytokines, and other chemicals, including lipidsinvolved in cell recruitment and migration, cell proliferation and thecontrol of lipid and protein synthesis.

For example, the expression of PDGF (platelet derived growth factor) orits receptor was studied: in rats during repair of arterial injury(Majesky et al., 1990, J. Cell Biol. 111: 2149); in adherent cultures ofhuman monocyte-derived macrophages treated with oxidized LDL (Malden etal., 1991, J. Biol. Chem. 266: 13901); and in bovine aortic endothelialcells subjected to fluid shear stress (Resnick et al., 1993, Proc. Natl.Acad. Sci. USA 90: 4591-4595). Expression of IGF-I (insulin-like growthfactor-I) was studied after balloon deendothelialization of rat aorta(Cercek et al., 1990, Circulation Research 66: 1755-1760) .

Other studies have focused on the expression of adhesion-molecules onthe surface of activated endothelial cells which mediate monocyteadhesion. These adhesion molecules include intracellular adhesionmolecule-1, ICAM-1 (Simmons et al., 1988, Nature, 331: 624-627), ELAM(Bevilacqua et al., 1989, Science 243: 1160-1165; Bevilacqua et al.,1991, Cell 67: 233), and vascular cell adhesion molecule, VCAM-1 (Osbornet al., 1989, Cell 59: 1203-1211); all of these surface molecules areinduced transcriptionally in the presence of IL-1. Histological studiesreveal that ICAM-1, ELAM and VCAM-1 are expressed on endothelial cellsin areas of lesion formation in vivo (Cybulsky et al., 1991, Science251: 788-791; 1991, Arterioscler. Thromb. 11: 1397a; Poston et al.,1992, Am. J. Pathol. 140: 665-673). VCAM-1 and ICAM-1 were shown to beinduced in cultured rabbit arterial endothelium, as well as in culturedhuman iliac artery endothelial cells by lysophophatidylcholine, a majorphospholipid component of atherogenic lipoproteins. (Kume et ale, 1992,J. Clin. Invest. 90: 1138-1144). VCAM-I, ICAM-1, and class II majorhistocompatibility antigens were reported to be induced in response toinjury to rabbit aorta (Tanaka, et al., 1993, Circulation 88:1788-1803).

Cytomegalovirus (CMV) has been implicated in restenosis as well asatherosclerosis in general (Speir, et ale, 1994, Science 265: 391-394).It was observed that the CMV protein IE84 apparently predisposes smoothmuscle cells to increased growth at the site of restenosis by combiningwith and inactivating p53 protein, which is known to suppress tumors inits active form.

The foregoing studies are aimed at defining the role of particular geneproducts presumed to be involved in the excessiveinflammatory-fibroproliferative response leading to atheroscleroticplaque formation. However, such approaches cannot identify the fullpanoply of gene products that are involved in the disease process, muchless identify those which may serve as therapeutic targets for thediagnosis and treatment of various forms of cardiovascular disease.

3. SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for thetreatment and diagnosis of cardiovascular disease, including but notlimited to, atherosclerosis, ischemia/reperfusion, hypertension,restenosis, and arterial inflammation. Specifically, genes areidentified and described which are differentially expressed incardiovascular disease states, relative to their expression in normal,or non-cardiovascular disease states.

"Differential expression", as used herein, refers to both quantitativeas well as qualitative differences in the genes' temporal and/or tissueexpression patterns Differentially expressed genes may represent"fingerprint genes," and/or "target genes." "Fingerprint gene," as usedherein, refers to a differentially expressed gene whose expressionpattern may be utilized as part of a prognostic or diagnosticcardiovascular disease evaluation, or which, alternatively, may be usedin methods for identifying compounds useful for the treatment ofcardiovascular disease "Target gene", as used herein, refers to adifferentially expressed gene involved in cardiovascular disease suchthat modulation of the level of target gene expression or of target geneproduct activity may act to ameliorate a cardiovascular diseasecondition. Compounds that modulate target gene expression or activity ofthe target gene product can be used in the treatment of cardiovasculardisease Further, "pathway genes" are defined via the ability of theirproducts to interact with other gene products involved in cardiovasculardisease. Pathway genes may also exhibit target gene and/or fingerprintgene characteristics. Although the genes described herein may bedifferentially expressed with respect to cardiovascular disease, and/ortheir products may interact with gene products important tocardiovascular disease, the genes may also be involved in mechanismsimportant to additional cardiovascular processes.

The invention includes the products of such fingerprint, target, andpathway genes, as well as antibodies to such gene products. Furthermore,the engineering and use of cell- and animal-based models ofcardiovascular disease to which such gene products may contribute arealso described.

The present invention encompasses methods for prognostic and diagnosticevaluation of cardiovascular disease conditions, and for theidentification of subjects exhibiting a predisposition to suchconditions. Furthermore, the invention provides methods for evaluatingthe efficacy of drugs, and monitoring the progress of patients, involvedin clinical trials for the treatment of cardiovascular disease.

The invention also provides methods for the identification of compoundsthat modulate the expression of genes or the activity of gene productsinvolved in cardiovascular disease, as well as methods for the treatmentof cardiovascular disease which may involve the administration of suchcompounds to individuals exhibiting cardiovascular disease symptoms ortendencies.

The invention is based, in part, on systematic search strategiesinvolving in vivo and in vitro cardiovascular disease paradigms coupledwith sensitive and high throughput gene expression assays. In contrastto approaches that merely evaluate the expression of a given geneproduct presumed to play a role in a disease process, the searchstrategies and assays used herein permit the identification of allgenes, whether known or novel, that are expressed or repressed in thedisease condition, as well as the evaluation of their temporalregulation and function during disease progression. This comprehensiveapproach and evaluation permits the discovery of novel genes and geneproducts, as well as the identification of an array of genes and geneproducts (whether novel or known) involved in novel pathways that play amajor role in the disease pathology. Thus, the invention allows one todefine targets useful for diagnosis, monitoring, rational drug screeningand design, and/or other therapeutic intervention.

In the working examples described herein, five novel human genes areidentified that are demonstrated to be differentially expressed indifferent cardiovascular disease states. The identification of thesegenes and the characterization of their expression in particular diseasestates provide newly identified roles in cardiovascular disease forthese genes.

Specifically, fchd531, fchd540, and fchd545 are novel genes that areeach differentially regulated in endothelial cells subjected to shearstress. fchd531 and fchd545 are each down-regulated, whereas fchd540 isup-regulated by shear stress. fchd602 and fchd605 are novel genes thatare each up-regulated in monocytes treated with oxidized LDL.Accordingly, methods are provided for the diagnosis, monitoring inclinical trials, screening for therapeutically effective compounds, andtreatment of cardiovascular disease based upon the discoveries hereinregarding the expression patterns of fchd531, fchd540, fchd545, fchd602,and fchd605.

The characteristic up-regulation of genes fchd540, fchd602, and fchd605can be used to design cardiovascular disease treatment strategies. Forthose up-regulated genes that have a causative effect on the diseaseconditions, treatment methods can be designed to reduce or eliminatetheir expression, particularly in endothelial cells or monocytes.Alternatively, treatment methods include inhibiting the activity of theprotein products of these genes. For those up-regulated genes that havea protective effect, treatment methods can be designed for enhancing theactivity of the products of such genes.

In either situation, detecting expression of these genes in excess ofnormal expression provides for the diagnosis of cardiovascular disease.Furthermore, in testing the efficacy of compounds during clinicaltrials, a decrease in the level of the expression of these genescorresponds to a return from a disease condition to a normal state, andthereby indicates a positive effect of the compound. The cardiovasculardiseases that may be so diagnosed, monitored in clinical trials, andtreated include but are not limited to atherosclerosis,ischemia/reperfusion, hypertension, restenosis, and arterialinflammation.

The characteristic down-regulation of fchd531 and fchd545 can also beused to design cardiovascular disease treatment strategies. For thosegenes whose down-regulation has a pathogenic effect, treatment methodscan be designed to restore or increase their expression, particularly inendothelial cells. Alternatively, treatment methods include increasingthe activity of the protein products of these genes. For those geneswhose down-regulation has a protective effect, treatment methods can bedesigned for decreasing the amount or activity of the products of suchgenes.

In either situation, detecting expression of these genes in below normalexpression provides for the diagnosis of cardiovascular disease.Furthermore, in testing the efficacy of compounds during clinicaltrials, an increase in the level of the expression of these genescorresponds to a return from a disease condition to a normal state, andthereby indicates a positive effect of the compound. The cardiovasculardiseases that may be so diagnosed, monitored in clinical trials, andtreated include but are not limited to atherosclerosis,ischemia/reperfusion, hypertension, restenosis, and arterialinflammation.

The invention encompasses methods for screening compounds and othersubstances for treating cardiovascular disease by assaying their abilityto modulate the expression of the target genes disclosed herein oractivity of the protein products of the target genes Such screeningmethods include, but are not limited to, assays for identifyingcompounds and other substances that interact with (e.g., bind to) thetarget gene protein products

In addition, the invention encompasses methods for treatingcardiovascular disease by administering compounds and other substancesthat modulate the overall activity of the target gene products.Compounds and other substances can effect such modulation either on thelevel of target gene expression or target protein activity.

The invention is based in part on the identification of novelprotein-protein interactions of the rchd534 protein with itself and withthe fchd540 protein, as well as interactions of the rchd534 protein orthe fchd540 protein with other protein members of the TGF-β signallingpathway. The rchd534 gene was described in Applicant's co-pendingapplication Ser. No. 08/485,573, filed Jun. 7, 1995, which is herebyincorporated by reference in its entirety. Screening methods areprovided for identifying compounds and other substances for treatingcardiovascular disease by assaying their ability to inhibit theseinteractions. Furthermore, methods are provided for identifyingcompounds and other substances that enhance the TGF-β response bymodulating the activity of the expression of the rchd534 or fchd540genes or the activity of their gene products. In addition, methods areprovided for treating cardiovascular disease by administering compoundsand other substances that inhibit these protein interactions.

The invention is based in part on the identification of the endothelialcell specific expression pattern of two genes, rchd534 and fchd540,whose protein products inhibit the TGF-β response. Accordingly, therchd534 and fchd540 genes may be targets for intervention in a varietyof inflammatory and fibroproliferative disorders that involveendothelial cells, including, but not limited to, cancer angiogenesis,inflammation, and fibrosis.

Membrane bound target gene products containing extracellular domains canbe a particularly useful target for treatment methods as well asdiagnostic and clinical monitoring methods. The fchd602 gene, forexample, encodes a transmembrane protein, which contains multipletransmembrane domains and, therefore, can be readily contacted by othercompounds on the cell surface. Accordingly, natural ligands, derivativesof natural ligands, and antibodies that bind to the fchd602 gene productcan be utilized to inhibit its activity, or alternatively, to target thespecific destruction of cells that express the gene. Furthermore, theextracellular domains of the fchd602 gene product provide targets whichallow for the design of especially efficient screening systems foridentifying compounds that bind to the fchd602 gene product.

Such an assay system can also be used to screen and identify antagonistsof the interaction between the fchd602 gene product and ligands thatbind to the fchd602 gene product. For example, the compounds can act asdecoys by binding to the endogenous (i.e., natural) ligand for thefchd602 gene product. The resulting reduction in the amount ofligand-bound fchd602 gene transmembrane protein will modulate theactivity of disease state cells, such as monocytes. Soluble proteins orpeptides, such as peptides comprising one or more of the extracellulardomains, or portions and/or analogs thereof of the fchd602 gene product,including, for example, soluble fusion proteins such as Ig-tailed fusionproteins, can be particularly useful for this purpose.

Similarly, antibodies that are specific to one or more of theextracellular domains of the fchd602 product provide for the readydetection of this target gene product in diagnostic tests or in clinicaltest monitoring Accordingly, endothelial cells can be treated, either invivo or in vitro, with such a labeled antibody to determine the diseasestate of endothelial cells. Because the fchd602 gene product isup-regulated in monocytes in the disease state, its detection positivelycorresponds with cardiovascular disease.

Such methods for treatment, diagnosis, and clinical test monitoringwhich use the fchd602 gene product as described above can also beapplied to other target genes that encode transmembrane gene products,including but not limited to the fchd545 gene, which encodes multipletransmembrane domains and extracellar domains.

The examples presented in Sections 6 and 7, below, demonstrate the useof the cardiovascular disease paradigms of the invention to identifycardiovascular disease target genes.

The example presented in Section 8, below, demonstrates the use offingerprint genes in diagnostics and as surrogate markers for testingthe efficacy of candidate drugs in basic research and in clinical trials

The example presented in Section 9, below, demonstrates the use offingerprint genes, particularly fchd545, in the imaging of a diseasedcardiovascular tissue

The example presented in Section 11, below, demonstrates the interactionof two target gene products, the rchd534 and fchd540 proteins, and thefurther characterization of their roles in cardiovascular disease andthe TGF-β signalling pathway.

4. DESCRIPTION OF THE FIGURES

FIGS. 1A-1C Nucleotide sequence (SEQ ID NO: 1) and encoded amino acidsequence (SEQ ID NO: 2) of the fchd531 gene.

FIGS. 2A-C Nucleotide sequence (SEQ ID NO: 3) and encoded amino acidsequence (SEQ ID NO: 4) of the fchd540 gene FIG. 3.

FIGS. 3A-3B. Nucleotide sequence (SEQ ID NO: 5) and encoded amino acidsequence (SEQ ID NO: 6) of the fchd545 gene.

FIG. 4. Nucleotide sequence (SEQ ID NO: 7) and encoded amino acidsequence (SEQ ID NO: 8) from the fchd602 gene.

FIG. 5. Nucleotide sequence (SEQ ID NO: 9) and encoded amino acidsequence (SEQ ID NO: 10) from the fchd605 gene.

FIG. 6A-6C Nucleotide sequence (SEQ ID NO: 11) and encoded amino acidsequence (SEQ ID NO: 12) of the rchd534 gene.

5. DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for the diagnosis and treatment ofcardiovascular disease, including but not limited to atherosclerosis,ischemia/reperfusion, hypertension, restenosis, and arterialinflammation, are described. The invention is based, in part, on theevaluation of the expression and role of all genes that aredifferentially expressed in paradigms that are physiologically relevantto the disease condition. This permits the definition of diseasepathways and the identification of targets in the pathway that areuseful both diagnostically and therapeutically.

Genes, termed "target genes" and/or "fingerprint genes" which aredifferentially expressed in cardiovascular disease conditions, relativeto their expression in normal, or non-cardiovascular disease conditions,are described in Section 5.4. Additionally, genes, termed "pathwaygenes" whose gene products exhibit an ability to interact with geneproducts involved in cardiovascular disease are also described inSection 5.4. Pathway genes may additionally have fingerprint and/ortarget gene characteristics. Methods for the identification of suchfingerprint, target, and pathway genes are described in Sections 5.1,5.2, and 5.3.

Further, the gene products of such fingerprint, target, and pathwaygenes are described in Section 5.4.2, antibodies to such gene productsare described in Section 5.4.3, as are cell- and animal-based models ofcardiovascular disease to which such gene products may contribute, inSection 5.4.4.

Methods for the identification of compounds which modulate theexpression of genes or the activity of gene products involved incardiovascular disease are described in Section 5.5. Methods formonitoring the efficacy of compounds during clinical trials aredescribed in Section 5.5.4. Additionally described below, in Section5.6, are methods for the treatment of cardiovascular disease.

Also discussed below, in Section 5.8, are methods for prognostic anddiagnostic evaluation of cardiovascular disease, including theidentification of subjects exhibiting a predisposition to this disease,and the imaging of cardiovascular disease conditions.

5.1. Identification of Differentially Expressed Genes

This section describes methods for the identification of genes which areinvolved in cardiovascular disease, including but not limited toatherosclerosis, ischemia/reperfusion, hypertension, restenosis, andarterial inflammation. Such genes may represent genes which aredifferentially expressed in cardiovascular disease conditions relativeto their expression in normal, or non-cardiovascular disease conditions.Such differentially expressed genes may represent "target" and/or"fingerprint" genes. Methods for the identification of suchdifferentially expressed genes are described, below, in this section.Methods for the further characterization of such differentiallyexpressed genes, and for their identification as target and/orfingerprint genes, are presented, below, in Section 5.3.

"Differential expression" as used herein refers to both quantitative aswell as qualitative differences in the genes' temporal and/or tissueexpression patterns. Thus, a differentially expressed gene may have itsexpression activated or completely inactivated in normal versuscardiovascular disease conditions (e.g., treated with oxidized LDLversus untreated), or under control versus experimental conditions Sucha qualitatively regulated gene -will exhibit an expression patternwithin a given tissue or cell type which is detectable in either controlor cardiovascular disease subjects, but is not detectable in both.Alternatively, such a qualitatively regulated gene will exhibit anexpression pattern within a given tissue or cell type which isdetectable in either control or experimental subjects, but is notdetectable in both. "Detectable", as used herein, refers to an RNAexpression pattern which is detectable via the standard techniques ofdifferential display, reverse transcriptase- (RT-) PCR and/or Northernanalyses, which are well known to those of skill in the art.

Alternatively, a differentially expressed gene may have its expressionmodulated, i.e., quantitatively increased or decreased, in normal versuscardiovascular disease states, or under control versus experimentalconditions. The degree to which expression differs in normal versuscardiovascular disease or control versus experimental states need onlybe large enough to be visualized via standard characterizationtechniques, such as, for example, the differential display techniquedescribed below. Other such standard characterization techniques bywhich expression differences may be visualized include but are notlimited to quantitative RT-PCR and Northern analyses.

Differentially expressed genes may be further described as target genesand/or fingerprint genes. "Fingerprint gene," as used herein, refers toa differentially expressed gene whose expression pattern may be utilizedas part of a prognostic or diagnostic cardiovascular disease evaluation,or which, alternatively, may be used in methods for identifyingcompounds useful for the treatment of cardiovascular disease. Afingerprint gene may also have the characteristics of a target gene.

"Target gene", as used herein, refers to a differentially expressed geneinvolved in cardiovascular disease in a manner by which modulation ofthe level of target gene expression or of target gene product activitymay act to ameliorate symptoms of cardiovascular disease. A target genemay also have the characteristics of a fingerprint gene.

A variety of methods may be utilized for the identification of geneswhich are involved in cardiovascular disease. These methods include butare not limited to the experimental paradigms described, below, inSection 5.1.1. Material from the paradigms may be characterized for thepresence of differentially expressed gene sequences as discussed, below,in Section 5.1.2.

5.1.1 Paradigms for the Identification of Differentially Expressed Genes

One strategy for identifying genes that are involved in cardiovasculardisease is to detect genes that are expressed differentially underconditions associated with the disease versus non-disease conditions Thesub-sections below describe a number of experimental systems, calledparadigms, which may be used to detect such differentially expressedgenes. In general, the paradigms include at least one experimentalcondition in which subjects or samples are treated in a mannerassociated with cardiovascular disease, in addition to at least oneexperimental control condition lacking such disease associated treatmentDifferentially expressed genes are detected, as described herein, below,by comparing the pattern of gene expression between the experimental andcontrol conditions.

Once a particular gene has been identified through the use of one suchparadigm, its expression pattern may be further characterized bystudying its expression in a different paradigm A gene may, for example,be regulated one way in a given paradigm (e.g., up-regulation), but maybe regulated differently in some other paradigm (e.g., down-regulation)Furthermore, while different genes may have similar expression patternsin one paradigm, their respective expression patterns may differ fromone another under a different paradigm Such use of multiple paradigmsmay be useful in distinguishing the roles and relative importance ofparticular genes in cardiovascular disease

5.1.1.1. Foam Cell Paradigm--1

Among the paradigms which may be utilized for the identification ofdifferentially expressed genes involved in atherosclerosis, for example,are paradigms designed to analyze those genes which may be involved infoam cell formation Such paradigms may serve to identify genes involvedin the differentiation of this cell type, or their uptake of oxidizedLDL.

One embodiment of such a paradigm, hereinafter referred to as ParadigmA, is carried out as follows: First, human blood is drawn and peripheralmonocytes are isolated by methods routinely practiced in the art. Thesehuman monocytes can then be used immediately or cultured in vitro, usingmethods routinely practiced in the art, for 5 to 9 days where theydevelop more macrophage-like characteristics such as the up-regulationof scavenger receptors These cells are then treated for various lengthsof time with agents thought to be involved in foam cell formation Theseagents include but are not limited to oxidized LDL, acetylated LDL,lysophosphatidylcholine, and homocysteine. Control monocytes that areuntreated or treated with native LDL are grown in parallel. At a certaintime after addition of the test agents, the cells are harvested andanalyzed for differential expression as described in detail in Section5.1.2., below. The Example presented in Section 6, below, demonstratesin detail the use of such a foam cell paradigm to identify genes whichare differentially expressed in treated versus control cells.

5.1.1.2. Cell Paradigm--2

Alternative paradigms involving monocytes for detecting differentiallyexpressed genes associated with atherosclerosis involve the simulationof the phenomenon of transmigration. When monocytes encounter arterialinjury, they adhere to the vascular endothelial layer, transmigrateacross this layer, and locate between the endothelium and the layer ofsmooth muscle cells that ring the artery This phenomenon can be mimickedin vitro by culturing a layer of endothelial cells isolated, forexample, from human umbilical cord. Once the endothelial monolayerforms, monocytes drawn from peripheral blood are cultured on top of theendothelium in the presence and absence of LDL. After several hours, themonocytes transmigrate through the endothelium and develop into foamcells after 3 to 5 days when exposed to LDL. In this system, as in vivo,the endothelial cells carry out the oxidation of LDL which is then takenup by the monocytes. As described in sub-section 5.1.2. below, thepattern of gene expression can then be compared between these foam cellsand untreated monocytes.

5.1.1.3. Foam Cell Paradigm--3

Yet another system includes the third cell type, smooth muscle cell,that plays a critical role in atherogenesis (Navab et al., 1988, J.Clin. Invest., 82: 1853). In this system, a multilayer of human aorticsmooth muscle cells was grown on a micropore filter covered with a gellayer of native collagen, and a monolayer of human aortic endothelialcells was grown on top of the collagen layer. Exposure of this cocultureto human monocytes in the presence of chemotactic factor rFHLP resultedin monocyte attachment to the endothelial cells followed by migrationacross the endothelial monolayer into the collagen layer of thesubendothelial space This type of culture can also be treated with LDLto generate foam cells. The foam cells can then be harvested and theirpattern of gene expression compared to that of untreated cells asexplained below in sub-section 5.1.2.

5.1.1.4. In Vivo Monocyte Paradigm

An alternative embodiment of such paradigms for the study of monocytes,hereinafter referred to as Paradigm B, involves differential treatmentof human subjects through the dietary control of lipid consumption Suchhuman subjects are held on a low fat/low cholesterol diet for threeweeks, at which time blood is drawn, monocytes are isolated according tothe methods routinely practiced in the art, and RNA is purified, asdescribed below, in sub-section 5.1.2. These same patients aresubsequently switched to a high fat/high cholesterol diet and monocyteRNA is purified again. The patients may also be fed a third, combinationdiet containing high fat/low cholesterol and monocyte RNA may bepurified once again. The order in which patients receive the diets maybe varied. The RNA derived from patients maintained on two of the diets,or on all three diets, may then be compared and analyzed fordifferential gene expression as, explained below in sub-section 5.1.2.

5.1.1.5. Endothelial Cell--IL-1 Paradigm

In addition to the detection of differential gene expression inmonocytes, paradigms focusing on endothelial cells may be used to detectgenes involved in cardiovascular disease. In one such paradigm,hereinafter referred to as Paradigm C, human umbilical vein endothelialcells (HUVEC's) are grown in vitro Experimental cultures are treatedwith human IL-1β, a factor known to be involved in the inflammatoryresponse, in order to mimic the physiologic conditions involved in theatherosclerotic state Alternatively experimental HUVEC cultures may betreated with lysophosphatidylcholine, a major phospholipid component ofatherogenic lipoproteins or oxidized human LDL. Control cultures aregrown in the absence of these compounds.

After a certain period of treatment, experimental and control cells areharvested and analyzed for differential gene expression as described insub-section 5.1.2, below.

5.1.1.6. Endothelial Cell--Shear Stress Paradigm

In another paradigm involving endothelial cells, hereinafter referred toas Paradigm D, cultures are exposed to fluid shear stress which isthought to be responsible for the prevalence of atherosclerotic lesionsin areas of unusual circulatory flow. Unusual blood flow also plays arole in the harmful effects of ischemia/reperfusion, wherein an organreceiving inadequate blood supply is suddenly reperfused with anoverabundance of blood when the obstruction is overcome.

Cultured HUVEC monolayers are exposed to laminar sheer stress byrotating the culture in a specialized apparatus containing liquidculture medium (Nagel et al., 1994, J. Clin. Invest. 94: 885-891).Static cultures grown in the same medium serve as controls. After acertain period of exposure to shear stress, experimental and controlcells are harvested and analyzed for differential gene expression asdescribed in sub-section 5.1.2, below. The Example presented in Section7, below, demonstrates the use of such a shear stressed endothelial cellparadigm to identify sequences which are differentially expressed inexposed versus control cells.

In all such paradigms designed to identify genes which are involved incardiovascular disease, including but not limited to those describedabove in Sections 5.1.1.1 through 5.1.1.6, compounds such as drugs knownto have an ameliorative effect on the disease symptoms may beincorporated into the experimental system Such compounds may includeknown therapeutics, as well as compounds that are not useful astherapeutics due to their harmful side effects. Test cells that arecultured as explained in the paradigms described in Sections 5.1.1.1through 5.1.1.6, for example, may be exposed to one of these compoundsand analyzed for differential gene expression with respect to untreatedcells, according to the methods described below in Section 5.1.2. Inprinciple, according to the particular paradigm, any cell type involvedin the disease may be treated at any stage of the disease process bythese compounds.

Test cells may also be compared to unrelated cells (e.g., fibroblasts)that are also treated with the compound, in order to screen out genericeffects on gene expression that might not be related to the disease.Such generic effects might be manifest by changes in gene expressionthat are common to the test cells and the unrelated cells upon treatmentwith the compound.

By these methods, the genes and gene products upon which these compoundsact can be identified and used in the assays described below to identifynovel therapeutic compounds for the treatment of cardiovascular disease.

5.1.2. Analysis of Paradigm Material

In order to identify differentially expressed genes, RNA, either totalor mRNA, may be isolated from one or more tissues of the subjectsutilized in paradigms such as those described earlier in this Section.RNA samples are obtained from tissues of experimental subjects and fromcorresponding tissues of control subjects. Any RNA isolation techniquewhich does not select against the isolation of mRNA may be utilized forthe purification of such RNA samples. See, for example, Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,N.Y.; and Ausubel, F. M. et al., eds., 1987-1993, Current Protocols inMolecular Biology, John Wiley & Sons, Inc. New York, both of which areincorporated herein by reference in their entirety Additionally, largenumbers of tissue samples may readily be processed using techniques wellknown to those of skill in the art, such as, for example, thesingle-step RNA isolation process of Chomczynski, P. (1989, U.S. Pat.No. 4,843,155), which is incorporated herein by reference in itsentirety

Transcripts within the collected RNA samples which represent RNAproduced by differentially expressed genes may be identified byutilizing a variety of methods which are well known to those of skill inthe art. For example, differential screening (Tedder, T. F. et al.,1988, Proc. Natl. Acad. Sci. USA 85:208-212), subtractive hybridization(Hedrick, S. M. et al., 1984, Nature 308:149-153; Lee, S. W. et al.,1984, Proc. Natl. Acad. Sci. USA 88:2825), and, preferably, differentialdisplay (Liang, P., and Pardee, A. B., 1993, U.S. Pat. No. 5,262,311,which is incorporated herein by reference in its entirety), may beutilized to identify nucleic acid sequences derived from genes that aredifferentially expressed.

Differential screening involves the duplicate screening of a cDNAlibrary in which one copy of the library is screened with a total cellcDNA probe corresponding to the mRNA population of one cell type while aduplicate copy of the cDNA library is screened with a total cDNA probecorresponding to the mRNA population of a second cell type. For example,one cDNA probe may correspond to a total cell cDNA probe of a cell typederived from a control subject, while the second cDNA probe maycorrespond to a total cell cDNA probe of the same cell type derived froman experimental subject. Those clones which hybridize to one probe butnot to the other potentially represent clones derived from genesdifferentially expressed in the cell type of interest in control versusexperimental subjects.

Subtractive hybridization techniques generally involve the isolation ofmRNA taken from two different sources, e.g., control and experimentaltissue, the hybridization of the mRNA or single-stranded cDNAreverse-transcribed from the isolated mRNA, and the removal of allhybridized, and therefore double-stranded, sequences. The remainingnon-hybridized, single-stranded cDNAs, potentially represent clonesderived from genes that are differentially expressed in the two mRNAsources. Such single-stranded cDNAs are then used as the startingmaterial for the construction of a library comprising clones derivedfrom differentially expressed genes.

The differential display technique describes a procedure, utilizing thewell known polymerase chain reaction (PCR; the experimental embodimentset forth in Hullis, K. B., 1987, U.S. Pat. No. 4,683,202) which allowsfor the identification of sequences derived from genes which aredifferentially expressed. First, isolated RNA is reverse-transcribedinto single-stranded cDNA, utilizing standard techniques which are wellknown to those of skill in the art. Primers for the reversetranscriptase reaction may include, but are not limited to, oligodT-containing primers, preferably of the reverse primer type ofoligonucleotide described below. Next, this technique uses pairs of PCRprimers, as described below, which allow for the amplification of clonesrepresenting a random subset of the RNA transcripts present within anygiven cell. Utilizing different pairs of primers allows each of the mRNAtranscripts present in a cell to be amplified. Among such amplifiedtranscripts may be identified those which have been produced fromdifferentially expressed genes.

The reverse oligonucleotide primer of the primer pairs may contain anoligo dT stretch of nucleotides, preferably eleven nucleotides long, atits 5' end, which hybridizes to the poly(A) tail of mRNA or to thecomplement of a cDNA reverse transcribed from an mRNA poly(A) tail.Second, in order to increase the specificity of the reverse primer, theprimer may contain one or more, preferably two, additional nucleotidesat its 3' end. Because, statistically, only a subset of the mRNA derivedsequences present in the sample of interest will hybridize to suchprimers, the additional nucleotides allow the primers to amplify only asubset of the mRNA derived sequences present in the sample of interest.This is preferred in that it allows more accurate and completevisualization and characterization of each of the bands representingamplified sequences.

The forward primer may contain a nucleotide sequence expected,statistically, to have the ability to hybridize to cDNA sequencesderived from the tissues of interest. The nucleotide sequence may be anarbitrary one, and the length of the forward oligonucleotide primer mayrange from about 9 to about 13 nucleotides, with about 10 nucleotidesbeing preferred. Arbitrary primer sequences cause the lengths of theamplified partial cDNAs produced to be variable, thus allowing differentclones to be separated by using standard denaturing sequencing gelelectrophoresis.

PCR reaction conditions should be chosen which optimize amplifiedproduct yield and specificity, and, additionally, produce amplifiedproducts of lengths which may be resolved utilizing standard gelelectrophoresis techniques. Such reaction conditions are well known tothose of skill in the art, and important reaction parameters include,for example, length and nucleotide sequence of oligonucleotide primersas discussed above, and annealing and elongation step temperatures andreaction times.

The pattern of clones resulting from the reverse transcription andamplification of the mRNA of two different cell types is displayed viasequencing gel electrophoresis and compared Differences in the twobanding patterns indicate potentially differentially expressed genes.

Once potentially differentially expressed gene sequences have beenidentified via bulk techniques such as, for example, those describedabove, the differential expression of such putatively differentiallyexpressed genes should be corroborated. Corroboration may beaccomplished via, for example, such well known techniques as Northernanalysis and/or RT-PCR.

Upon corroboration, the differentially expressed genes may be furthercharacterized, and may be identified as target and/or fingerprint genes,as discussed, below, in Section 5.3.

Also, amplified sequences of differentially expressed genes obtainedthrough, for example, differential display may be used to isolate fulllength clones of the corresponding gene. The full length coding portionof the gene may readily be isolated, without undue experimentation, bymolecular biological techniques well known in the art. For example, theisolated differentially expressed amplified fragment may be labeled andused to screen a cDNA library. Alternatively, the labeled fragment maybe used to screen a genomic library.

PCR technology may also be utilized to isolate full length cDNAsequences. As described, above, in this Section, the isolated, amplifiedgene fragments obtained through differential display have 5' terminalends at some random point within the gene and have 3' terminal ends at aposition preferably corresponding to the 3' end of the transcribedportion of the gene. Once nucleotide sequence information from anamplified fragment is obtained, the remainder of the gene (i.e., the 5'end of the gene, when utilizing differential display) may be obtainedusing, for example, RT-PCR.

In one embodiment of such a procedure for the identification and cloningof full length gene sequences, RNA may be isolated, following standardprocedures, from an appropriate tissue or cellular source. A reversetranscription reaction may then be performed on the RNA using anoligonucleotide primer complimentary to the mRNA that corresponds to theamplified fragment, for the priming of first strand synthesis. Becausethe primer is anti-parallel to the mRNA, extension will proceed towardthe 5' end of the mRNA. The resulting RNA/DNA hybrid may then be"tailed" with guanines using a standard terminal transferase reaction,the hybrid may be digested with RNAase H, and second strand synthesismay then be primed with a poly-C primer. Using the two primers, the 5'portion of the gene is amplified using PCR. Sequences obtained may thenbe isolated and recombined with previously isolated sequences togenerate a full-length cDNA of the differentially expressed genes of theinvention For a review of cloning strategies and recombinant DNAtechniques, see e.g., Sambrook et al., 1989, supra; and Ausubel et al.,1989, supra.

5.2. Identification of Pathway Genes

This section describes methods for the identification of genes, termed"pathway genes", involved in cardiovascular disease "Pathway gene", asused herein, refers to a gene whose gene product exhibits the ability tointeract with gene products involved in cardiovascular disease A pathwaygene may be differentially expressed and, therefore, may additionallyhave the characteristics of a target and/or fingerprint gene.

Any method suitable for detecting protein-protein interactions may beemployed for identifying pathway gene products by identifyinginteractions between gene products and gene products known to beinvolved in cardiovascular disease. Such known gene products may becellular or extracellular proteins. Those gene products which interactwith such known gene products represent pathway gene products and thegenes which encode them represent pathway genes

Among the traditional methods which may be employed areco-immunoprecipitation, crosslinking and co-purification throughgradients or chromatographic columns. Utilizing procedures such as theseallows for the identification of pathway gene products. Once identified,a pathway gene product may be used, in conjunction with standardtechniques, to identify its corresponding pathway gene. For example, atleast a portion of the amino acid sequence of the pathway gene productmay be ascertained using techniques well known to those of skill in theart, such as via the Edman degradation technique (see, e.g., Creighton,1983, Proteins: Structures and Molecular Principles, W. H. Freeman &Co., N.Y. pp.34-49). The amino acid sequence obtained may be used as aguide for the generation of oligonucleotide mixtures that can be used toscreen for pathway gene sequences. Screening may be accomplished, forexample by standard hybridization or PCR technique. Techniques for thegeneration of oligonucleotide mixtures and screening are well-known.(See, e.g., Ausubel, supra., and PCR Protocols: A Guide to Methods andApplications, 1990, Innis, M. et al., eds. Academic Press, Inc., NewYork).

Additionally, methods may be employed which result in the simultaneousidentification of pathway genes which encode the protein interactingwith a protein involved in cardiovascular disease. These methodsinclude, for example, probing expression libraries with labeled proteinknown or suggested to be involved in cardiovascular disease, using thisprotein in a manner similar to the well known technique of antibodyprobing of λgt11 libraries

One such method which detects protein interactions in vivo, thetwo-hybrid system, is described in detail for illustration only and notby way of limitation. One version of this system has been described(Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and iscommercially available from Clontech (Palo Alto, Calif.)

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins: one consists of the DNA-binding domain of atranscription activator protein fused to a known protein, and the otherconsists of the activator protein's activation domain fused to anunknown protein that is encoded by a cDNA which has been recombined intothis plasmid as part of a cDNA library. The plasmids are transformedinto a strain of the yeast Saccharomyces cerevisiae that contains areporter gene (e.g., lacZ) whose regulatory region contains theactivator's binding sites. Either hybrid protein alone cannot activatetranscription of the reporter gene; the DNA-binding domain hybrid,because it does not provide activation function and the activationdomain hybrid, because it cannot localize to the activator's bindingsites. Interaction of the two proteins reconstitutes the functionalactivator protein and results in expression of the reporter gene, whichis detected by an assay for the reporter gene product.

The two-hybrid system or related methodology may be used to screenactivation domain libraries for proteins that interact with a known"bait" gene protein. Total genomic or cDNA sequences may be fused to theDNA encoding an activation domain. Such a library and a plasmid encodinga hybrid of the bait gene protein fused to the DNA-binding domain may becotransformed into a yeast reporter strain, and the resultingtransformants may be screened for those that express the reporter gene.These colonies may be purified and the library plasmids responsible forreporter gene expression may be isolated DNA sequencing may then be usedto identify the proteins encoded by the library plasmids.

For example, and not by way of limitation, the bait gene may be clonedinto a vector such that it is translationally fused to the DNA encodingthe DNA-binding domain of the GAL4 protein. Also by way of example, forthe isolation of genes involved in cardiovascular disease, previouslyisolated genes known or suggested to play a part in cardiovasculardisease may be used as the bait genes These include but are not limitedto the genes for bFGF, IGF-I, VEGF, IL-1, M-CSF, TGFβ, TGFα, TNFα,HB-EGF, PDGF, IFN-γ, and GM-CSF, to name a few.

A cDNA library of the cell line from which proteins that interact withbait gene are to be detected can be made using methods routinelypracticed in the art. According to the particular system describedherein, for example, the cDNA fragments may be inserted into a vectorsuch that they are translationally fused to the activation domain ofGAL4. This library may be co-transformed along with the bait gene-GAL4fusion plasmid into a yeast strain which contains a lacZ gene driven bya promoter which contains the GAL4 activation sequence A cDNA encodedprotein, fused to the GAL4 activation domain, that interacts with baitgene will reconstitute an active GAL4 protein and thereby driveexpression of the lacZ gene. Colonies which express lacZ may be detectedby their blue color in the presence of X-gal. The cDNA may then bepurified from these strains, and used to produce and isolate the baitgene-interacting protein using techniques routinely practiced in theart.

Once a pathway gene has been identified and isolated, it may be furthercharacterized as, for example, discussed below, in Section 5.3.

A preferred embodiment of the use of the yeast two-hybrid system isdescribed in detail in the example in Section 12, below. As described inSection 12, the yeast two-hybrid system was used to detect theinteraction between the protein products of two target genes, rchd534and fchd540.

5.3. Characterization of Differentially Expressed and Pathway Genes

Differentially expressed genes, such as those identified via the methodsdiscussed, above, in Section 5.1.1, pathway genes, such as thoseidentified via the methods discussed, above, in Section 5.2, as well asgenes identified by alternative means, may be further characterized byutilizing, for example, methods such as those discussed herein. Suchgenes will be referred to herein as "identified genes".

Analyses such as those described herein will yield information regardingthe biological function of the identified genes. An assessment of thebiological function of the differentially expressed genes, in addition,will allow for their designation as target and/or fingerprint genes.Specifically, any of the differentially expressed genes whose furthercharacterization indicates that a modulation of the gene's expression ora modulation of the gene product's activity may amelioratecardiovascular disease will be designated "target genes", as defined,above, in Section 5.1. Such target genes and target gene products, alongwith those discussed below, will constitute the focus of the compounddiscovery strategies discussed, below, in Section 5.5.

Any of the differentially expressed genes whose further characterizationindicates that such modulations may not positively affect cardiovasculardisease, but whose expression pattern contributes to a gene expression"fingerprint pattern" correlative of, for example, a cardiovasculardisease condition will be designated a "fingerprint gene". "Fingerprintpatterns" will be more fully discussed, below, in Section 5.8. It shouldbe noted that each of the target genes may also function as fingerprintgenes, as may all or a subset of the pathway genes.

It should further be noted that the pathway genes may also becharacterized according to techniques such as those described herein.Those pathway genes which yield information indicating that they aredifferentially expressed and that modulation of the gene's expression ora modulation of the gene product's activity may amelioratecardiovascular disease will be also be designated "target genes". Suchtarget genes and target gene products, along with those discussed above,will constitute the focus of the compound discovery strategiesdiscussed, below, in Section 5.5

It should be additionally noted that the characterization of one or moreof the pathway genes may reveal a lack of differential expression, butevidence that modulation of the gene's activity or expression may,nonetheless, ameliorate cardiovascular disease symptoms. In such cases,these genes and gene products would also be considered a focus of thecompound discovery strategies of Section 5.5, below.

In instances wherein a pathway gene's characterization indicates thatmodulation of gene expression or gene product activity may notpositively affect cardiovascular disease, but whose expression isdifferentially expressed and which contributes to a gene expressionfingerprint pattern correlative of, for example, a cardiovasculardisease state, such pathway genes may additionally be designated asfingerprint genes.

Among the techniques whereby the identified genes may be furthercharacterized, the nucleotide sequence of the identified genes, whichmay be obtained by utilizing standard techniques well known to those ofskill in the art, may be used to further characterize such genes. Forexample, the sequence of the identified genes may reveal homologies toone or more known sequence motifs which may yield information regardingthe biological function of the identified gene product.

Second, an analysis of the tissue distribution of the mRNA produced bythe identified genes may be conducted, utilizing standard techniqueswell known to those of skill in the art. Such techniques may include,for example, Northern analyses and RT-PCR. Such analyses provideinformation as to whether the identified genes are expressed in tissuesexpected to contribute to cardiovascular disease Such analyses may alsoprovide quantitative information regarding steady state mRNA regulation,yielding data concerning which of the identified genes exhibits a highlevel of regulation in, preferably, tissues which may be expected tocontribute to cardiovascular disease.

Such analyses may also be performed on an isolated cell population of aparticular cell type derived from a given tissue. Additionally, standardin situ hybridization techniques may be utilized to provide informationregarding which cells within a given tissue express the identified gene.Such analyses may provide information regarding the biological functionof an identified gene relative to cardiovascular disease in instanceswherein only a subset of the cells within the tissue is thought to berelevant to cardiovascular disease.

Third, the sequences of the identified genes may be used, utilizingstandard techniques, to place the genes onto genetic maps, e.g., mouse(Copeland & Jenkins, 1991, Trends in Genetics 7: 113-118) and humangenetic maps (Cohen, et al., 1993, Nature 366: 698-701). Such mappinginformation may yield information regarding the genes' importance tohuman disease by, for example, identifying genes which map near geneticregions to which known genetic cardiovascular disease tendencies map.

Fourth, the biological function of the identified genes may be moredirectly assessed by utilizing relevant in vivo and in vitro systems. Invivo systems may include, but are not limited to, animal systems whichnaturally exhibit cardiovascular disease predisposition, or ones whichhave been engineered to exhibit such symptoms, including but not limitedto the apoE-deficient atherosclerosis mouse model (Plump et al., 1992,Cell 71: 343-353). Such systems are discussed in Section 5.4.4.1, below.

In vitro systems may include, but are not limited to, cell-based systemscomprising cell types known or suspected of involvement incardiovascular disease. Such systems are discussed in detail, below, inSection 5.4.4.2.

In further characterizing the biological function of the identifiedgenes, the expression of these genes may be modulated within the in vivoand/or in vitro systems, i.e., either over- or underexpressed, and thesubsequent effect on the system then assayed. Alternatively, theactivity of the product of the identified gene may be modulated byeither increasing or decreasing the level of activity in the in vivoand/or in vitro system of interest, and its subsequent effect thenassayed.

The information obtained through such characterizations may suggestrelevant methods for the treatment of cardiovascular disease involvingthe gene of interest. For example, treatment may include a modulation ofgene expression and/or gene product activity. Characterizationprocedures such as those described herein may indicate where suchmodulation should involve an increase or a decrease in the expression oractivity of the gene or gene product of interest.

For example, genes which are up-regulated under disease conditions maybe involved in causing or exacerbating the disease condition. Treatmentsdirected at down-regulating the activity of such harmfully expressedgenes will ameliorate the disease condition. On the other hand, theup-regulation of genes under disease conditions may be part of aprotective response by affected cells. Treatments directed at increasingor enhancing the activity of such up-regulated gene products, especiallyin individuals lacking normal up-regulation, will similarly amelioratedisease conditions. Such methods of treatment are discussed, below, inSection 5.6.

5.4. Differentially Expressed and Pathway Genes

Identified genes, which include but are not limited to differentiallyexpressed genes such as those identified in Section 5.1.1, above, andpathway genes, such as those identified in Section 5.2, above, aredescribed herein. Specifically, the nucleic acid sequences and geneproducts of such identified genes are described herein. Further,antibodies directed against the identified genes' products, and cell-and animal-based models by which the identified genes may be furthercharacterized and utilized are also discussed in this Section.

5.4.1. Differentially Expressed and Pathway Gene Sequences

The differentially expressed and pathway genes of the invention arelisted below, in Table 1. Differentially expressed and pathway genenucleotide sequences are shown in FIGS. 1A-1C, 2A-2C, 3A-3B, 4, and 5.

Table 1 lists differentially expressed genes identified through, forexample, the paradigms discussed, above, in Section 5.1.1, and below, inthe examples presented in Sections 6 through 9. Table 1 also summarizesinformation regarding the further characterization of such genes.

First, the paradigm used initially to detect the differentiallyexpressed gene is described under the column headed "Paradigm ofOriginal Detection". The expression patterns of those genes which havebeen shown to be differentially expressed, for example, under one ormore of the paradigm conditions described in Section 5.1.1. aresummarized under the column headed "Paradigm Expression Pattern". Foreach of the tested genes, the paradigm which was used and the differencein the expression of the gene among the samples generated is shown. "↑"indicates that gene expression is up-regulated (i.e., there is anincrease in the amount of detectable mRNA) among the samples generated,while "↓" indicates that gene expression is down-regulated (i.e., thereis a decrease in the amount of detectable mRNA) among the samplesgenerated. "Detectable" as used herein, refers to levels of mRNA whichare detectable via, for example, standard Northern and/or RT-PCRtechniques which are well known to those of skill in the art.

Cell types in which differential expression was detected are alsosummarized in Table 1 under the column headed "Cell Type Detected in".The column headed "Chromosomal Location" provides the human chromosomenumber on which the gene is located. Additionally, in instances whereinthe genes contain nucleotide sequences similar or homologous tosequences found in nucleic acid databases, references to suchsimilarities are listed.

The genes listed in Table 1 may be obtained using cloning methods wellknown to those skilled in the art, including but not limited to the useof appropriate probes to detect the genes within an appropriate cDNA orgDNA (genomic DNA) library. (See, for example, Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories,which is incorporated by reference herein in its entirety). Probes forthe novel sequences reported herein may be obtained directly from theisolated clones deposited with the ATCC, as indicated in Table 2, below.Alternatively, oligonucleotide probes for the novel genes may besynthesized based on the DNA sequences disclosed herein in FIGS. 1-5.

The sequence obtained from clones containing partial coding sequences ornon-coding sequences can be used to obtain the entire coding region byusing the RACE method (Chenchik, et al., 1995, CLONTECHniques (X) 1:5-8; Barnes, 1994, Proc. Natl. Acad. Sci. USA 91: 2216-2220; and Chenget al., Proc. Natl Acad. Sci. USA 91: 5695-5699). oligonucleotides canbe designed based on the sequence obtained from the partial clone thatcan amplify a reverse transcribed mRNA encoding the entire codingsequence

Alternatively, probes can be used to screen cDNA libraries prepared froman appropriate cell or cell line in which the gene is transcribed Forexample, the genes described herein that were detected in monocytes maybe cloned from a cDNA library prepared from monocytes isolated asdescribed in Section 6.1.1, below.

The genes described herein that were detected in endothelial cells mayalso be cloned from a cDNA library constructed from endothelial cellsisolated as described in Progress in Hemostasis and Thrombosis, Vol. 3,P. Spaet, editor, Grune & Stratton Inc., New York, 1-28. Alternatively,the genes may be retrieved from a human placenta cDNA library (ClontechLaboratories, Palo Alto, Calif.), according to Takahashi et al., 1990,supra; a HUVEC cDNA library as described in Jones et al. 1993, supra; oran acute lymphoblastic leukemia (SUP-B2) cDNA library as described inCleary et al., 1986, supra, for example. Genomic DNA libraries can beprepared from any source.

                                      TABLE 1                                     __________________________________________________________________________    Differentially Expressed and Pathway Genes                                             Paradigm of                                                                            Paradigm                                                                            Cell Type                                               Gene Seq. ID #  Original Detection Expr. Pattern Detected in Ref            __________________________________________________________________________                                      Seq.                                        fchd531                                                                           1    D (Sectio  5.1.1.6)                                                                          Endothelial                                                                         New, 1                                                                            FIG. 1                                        fchd540 3 D   Endothelial New, 2 FIG. 2                                       fchd545 5 D   Endothelial New, 3 FIG. 3                                       fchd602 7 A (Section 5.1.1.1)   Monocytes New, 4 FIG. 4                       fchd605 9 A   Monocytes New, 5 FIG. 5                                       __________________________________________________________________________     1. GenBank accession number U05343.                                           2. Drosobila Mothers against dpp (Mad), Sekelsky et al., 1995, Genetics       139: 1347-1358.                                                               3. Human Voltagedependent Anion Channel, BlachlyDyson, E., et al., 1993,      J. Biol. Chem. 268: 1835-1841; and EST T24012                                 4. Rat C1-6, Diamond, R.H., et al., 1993, J. Biol. Chem. 268: 15185-15192     5. Mouse gly96, Charles, C.H., et al., 1993, Oncogene 8: 797-801; and EST     T49532.                                                                  

Table 2, below, lists the strains of E. coli deposited with the ATCCthat contain plasmids bearing the novel genes listed in Table 1.

                  TABLE 2                                                         ______________________________________                                                    Strain Deposited                                                    GENE with ATCC                                                              ______________________________________                                        fchd531     pFCHD531                                                            fchd540 pFCHD540                                                              fchd545 fchd545                                                             ______________________________________                                    

As used herein, "differentially expressed gene" (i.e. target andfingerprint gene) or "pathway gene" refers to (a) a gene containing atleast one of the DNA sequences disclosed herein (as shown in FIGS.1A-1C, 2A-2C, 3A-3B, 4 and 5), or contained in the clones listed inTable 2, as deposited with the ATCC; (b) any DNA sequence that encodesthe amino acid sequence encoded by the DNA sequences disclosed herein(as shown in FIGS. 1A-1C, 2A-2C, 3A-3B, 4, and 5), contained in theclones, listed in Table 2, as deposited with the ATCC or containedwithin the coding region of the gene to which the DNA sequencesdisclosed herein (as shown in FIGS. 1A-1C, 2A-2C, 3A-3B, 4, and 5) orcontained in the clones listed in Table 2, as deposited with the ATCC,belong; (c) any DNA sequence that hybridizes to the complement of thecoding sequences disclosed herein, contained in the clones listed inTable 2, as deposited with the ATCC, or contained within the codingregion of the gene to which the DNA sequences disclosed herein (as shownin FIGS. 1A-1C, 2A-2C, 3A-3B, 4, and 5) or contained in the cloneslisted in Table 2, as deposited with the ATCC, belong, under highlystringent conditions, e.g., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3)and encodes a gene product functionally equivalent to a gene productencoded by sequences contained within the clones listed in Table 2;and/or (d) any DNA sequence that hybridizes to the complement of thecoding sequences disclosed herein, (as shown in FIGS. 1A-1C, 2A-2C,3A-3B, 4, and 5) contained in the clones listed in Table 2, as depositedwith the ATCC or contained within the coding region of the gene to whichDNA sequences disclosed herein (as shown in FIGS. 1A-1C, 2A-2C, 3A-3B, 4and 5) or contained in the clones, listed in Table 2, as deposited withthe ATCC, belong, under less stringent conditions, such as moderatelystringent conditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C.(Ausubel et al., 1989, supra), yet which still encodes a functionallyequivalent gene product.

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, theDNA sequences (a) through (c), in the preceding paragraph. Suchhybridization conditions may be highly stringent or less highlystringent, as described above. In instances wherein the nucleic acidmolecules are deoxyoligonucleotides ("oligos"), highly stringentconditions may refer, e.g., to washing in 6×SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules may act as target gene antisense molecules,useful, for example, in target gene regulation and/or as antisenseprimers in amplification reactions of target gene nucleic acidsequences. Further, such sequences may be used as part of ribozymeand/or triple helix sequences, also useful for target gene regulation.Still further, such molecules may be used as components of diagnosticmethods whereby the presence of a cardiovascular disease-causing allele,may be detected

The invention also encompasses (a) DNA vectors that contain any of theforegoing coding sequences and/or their complements (i.e., antisense);(b) DNA expression vectors that contain any of the foregoing codingsequences operatively associated with a regulatory element that directsthe expression of the coding sequences; and (c) genetically engineeredhost cells that contain any of the foregoing coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Theinvention includes fragments of any of the DNA sequences disclosedherein.

In addition to the gene sequences described above, homologues of suchsequences, as may, for example be present in other species, may beidentified and may be readily isolated, without undue experimentation,by molecular biological techniques well known in the art. Further, theremay exist genes at other genetic loci within the genome that encodeproteins which have extensive homology to one or more domains of suchgene products. These genes may also be identified via similartechniques.

For example, the isolated differentially expressed gene sequence may belabeled and used to screen a cDNA library constructed from mRNA obtainedfrom the organism of interest. Hybridization conditions will be of alower stringency when the cDNA library was derived from an organismdifferent from the type of organism from which the labeled sequence wasderived. Alternatively, the labeled fragment may be used to screen agenomic library derived from the organism of interest, again, usingappropriately stringent conditions. Such low stringency conditions willbe well known to those of skill in the art, and will vary predictablydepending on the specific organisms from which the library and thelabeled sequences are derived. For guidance regarding such conditionssee, for example, Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989,Current Protocols in Molecular Biology, Green Publishing Associates andWiley Interscience, N.Y.

Further, a previously unknown differentially expressed or pathwaygene-type sequence may be isolated by performing PCR using twodegenerate oligonucleotide primer pools designed on the basis of aminoacid sequences within the gene of interest. The template for thereaction may be cDNA obtained by reverse transcription of mRNA preparedfrom human or non-human cell lines or tissue known or suspected toexpress a differentially expressed or pathway gene allele.

The PCR product may be subcloned and sequenced to insure that theamplified sequences represent the sequences of a differentiallyexpressed or pathway gene-like nucleic acid sequence. The PCR fragmentmay then be used to isolate a full length cDNA clone by a variety ofmethods. For example, the amplified fragment may be labeled and used toscreen a bacteriophage cDNA library. Alternatively, the labeled fragmentmay be used to screen a genomic library.

PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source. A reversetranscription reaction may be performed on the RNA using anoligonucleotide primer specific for the most 5' end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid may then be "tailed" with guanines using a standardterminal transferase reaction, the hybrid may be digested with RNAase H,and second strand synthesis may then be primed with a poly-C primer.Thus, cDNA sequences upstream of the amplified fragment may easily beisolated. For a review of cloning strategies which may be used, seee.g., Sambrook et al., 1989, supra.

In cases where the differentially expressed or pathway gene identifiedis the normal, or wild type, gene, this gene may be used to isolatemutant alleles of the gene. Such an isolation is preferable in processesand disorders which are known or suspected to have a genetic basis.Mutant alleles may be isolated from individuals either known orsuspected to have a genotype which contributes to cardiovascular diseasesymptoms. Mutant alleles and mutant allele products may then be utilizedin the therapeutic and diagnostic assay systems described below.

A cDNA of the mutant gene may be isolated, for example, by using PCR, atechnique which is well known to those of skill in the art. In thiscase, the first cDNA strand may be synthesized by hybridizing anoligo-dT oligonucleotide to mRNA isolated from tissue known or suspectedto be expressed in an individual putatively carrying the mutant allele,and by extending the new strand with reverse transcriptase. The secondstrand of the cDNA is then synthesized using an oligonucleotide thathybridizes specifically to the 5' end of the normal gene. Using thesetwo primers, the product is then amplified via PCR, cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell known to those of skill in the art. By comparing the DNA sequenceof the mutant gene to that of the normal gene, the mutation(s)responsible for the loss or alteration of function of the mutant geneproduct can be ascertained

Alternatively, a genomic or cDNA library can be constructed and screenedusing DNA or RNA, respectively, from a tissue known to or suspected ofexpressing the gene of interest in an individual suspected of or knownto carry the mutant allele. The normal gene or any suitable fragmentthereof may then be labeled and used as a probed to identify thecorresponding mutant allele in the library. The clone containing thisgene may then be purified through methods routinely practiced in theart, and subjected to sequence analysis as described, above, in thisSection.

Additionally, an expression library can be constructed utilizing DNAisolated from or cDNA synthesized from a tissue known to or suspected ofexpressing the gene of interest in an individual suspected of or knownto carry the mutant allele. In this manner, gene products made by theputatively mutant tissue may be expressed and screened using standardantibody screening techniques in conjunction with antibodies raisedagainst the normal gene product, as described, below, in Section 5.4.3.(For screening techniques, see, for example, Harlow, E. and Lane, eds.,1988, "Antibodies, A Laboratory Manual", Cold Spring Harbor Press, ColdSpring Harbors) In cases where the mutation results in an expressed geneproduct with altered function (e.g., as a result of a missensemutation), a polyclonal set of antibodies are likely to cross-react withthe mutant gene product. Library clones detected via their reaction withsuch labeled antibodies can be purified and subjected to sequenceanalysis as described in this Section, above.

5.4.2. Differentially Expressed and Pathway Gene Products

Differentially expressed and pathway gene products include thoseproteins encoded by the differentially expressed and pathway genesequences described in Section 5.4.1, above. Specifically,differentially expressed and pathway gene products may includedifferentially expressed and pathway gene polypeptides encoded by thedifferentially expressed and pathway gene sequences contained in theclones listed in Table 2, above, as deposited with the ATCC, orcontained in the coding regions of the genes to which DNA sequencesdisclosed herein (in FIGS. 1A-1C, 2A-2C, 3A-3B, 4, and 5) or containedin the clones, listed in Table 2, as deposited with the ATCC, belong,for example.

In addition, differentially expressed and pathway gene products mayinclude proteins that represent functionally equivalent gene products.Such an equivalent differentially expressed or pathway gene product maycontain deletions, additions or substitutions of amino acid residueswithin the amino acid sequence encoded by the differentially expressedor pathway gene sequences described, above, in Section 5.4.1, but whichresult in a silent change, thus producing a functionally equivalentdifferentially expressed on pathway gene product. Amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved.

For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. "Functionally equivalent", as utilized herein, refers toa protein capable of exhibiting a substantially similar in vivo activityas the endogenous differentially expressed or pathway gene productsencoded by the differentially expressed or pathway gene sequencesdescribed in Section 5.4.1, above. Alternatively, when utilized as partof assays such as those described, below, in Section 5.5, "functionallyequivalent" may refer to peptides capable of interacting with othercellular or extracellular molecules in a manner substantially similar tothe way in which the corresponding portion of the endogenousdifferentially expressed or pathway gene product would.

The differentially expressed or pathway gene products may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing the differentially expressed or pathway genepolypeptides and peptides of the invention by expressing nucleic acidencoding differentially expressed or pathway gene sequences aredescribed herein. Methods which are well known to those skilled in theart can be used to construct expression vectors containingdifferentially expressed or pathway gene protein coding sequences andappropriate transcriptional/translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. See, forexample, the techniques described in Sambrook et al., 1989, supra, andAusubel et al., 1989, supra. Alternatively, RNA capable of encodingdifferentially expressed or pathway gene protein sequences may bechemically synthesized using, for example, synthesizers See, forexample, the techniques described in "Oligonucleotide Synthesis", 1984,Gait, M. J. ed., IRL Press, Oxford, which is incorporated by referenceherein in its entirety

A variety of host-expression vector systems may be utilized to expressthe differentially expressed or pathway gene coding sequences of theinvention. Such host-expression systems represent vehicles by which thecoding sequences of interest may be produced and subsequently purified,but also represent cells which may, when transformed or transfected withthe appropriate nucleotide coding sequences, exhibit the differentiallyexpressed or pathway gene protein of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing differentiallyexpressed or pathway gene protein coding sequences; yeast (e.g.Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing the differentially expressed or pathway gene proteincoding sequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the differentiallyexpressed or pathway gene protein coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingdifferentially expressed or pathway gene protein coding sequences; ormammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5 K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for thedifferentially expressed or pathway gene protein being expressed. Forexample, when a large quantity of such a protein is to be produced, forthe generation of antibodies or to screen peptide libraries, forexample, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which thedifferentially expressed or pathway gene protein coding sequence may beligated individually into the vector in frame with the lac Z codingregion so that a fusion protein is produced; pIN vectors (Inouye &Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the likes pGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene protein can bereleased from the GST moiety.

In a preferred embodiment, full length cDNA sequences are appended within-frame Bam HI sites at the amino terminus and Eco RI sites at thecarboxyl terminus using standard PCR methodologies (Innis et al., 1990,supra) and ligated into the pGEX-2TK vector (Pharmacia, Uppsala,Sweden). The resulting cDNA construct contains a kinase recognition siteat the amino terminus for radioactive labelling and glutathioneS-transferase sequences at the carboxyl terminus for affinitypurification (Nilsson, et al., 1985, EMBO J. 4: 1075; Zabeau andStanley, 1982, EMBO J. 1: 1217.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The differentially expressed or pathwaygene coding sequence may be cloned individually into non-essentialregions (for example the polyhedrin gene) of the virus and placed undercontrol of an AcNPV promoter (for example the polyhedrin promoter).Successful insertion of differentially expressed or pathway gene codingsequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed. (E.g., see Smith et al., 1983, J. Virol. 46:584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the differentially expressed or pathway gene coding sequence ofinterest may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing differentially expressedor pathway gene protein in infected hosts. (E.g., See Logan & Shenk,1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiationsignals may also be required for efficient translation of inserteddifferentially expressed or pathway gene coding sequences. These signalsinclude the ATG initiation codon and adjacent sequences. In cases wherean entire differentially expressed or pathway gene, including its owninitiation codon and adjacent sequences, is inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only a portion of thedifferentially expressed or pathway gene coding sequence is inserted,exogenous translational control signals, including, perhaps, the ATGinitiation codon, must be provided. Furthermore, the initiation codonmust be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153:516-544).

In a preferred embodiment, cDNA sequences encoding the full-length openreading frames are ligated into pCMVβ replacing the β-galactosidase genesuch that cDNA expression is driven by the CHV promoter (Alam, 1990,Anal. Biochem. 188: 245-254; MacGregor & Caskey, 1989, Nucl. Acids Res.17: 2365; Norton & Corrin, 1985, Mol. Cell. Biol. 5: 281).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe differentially expressed or pathway gene protein may be engineered.Rather than using expression vectors which contain viral origins ofreplication, host cells can be transformed with DNA controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell lineswhich express the differentially expressed or pathway gene protein. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that affect the endogenous activity of thedifferentially expressed or pathway gene protein.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, etal., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre, et al., 1984, Gene 30:147) genes.

An alternative fusion protein system allows for the ready purificationof non-denatured fusion proteins expressed in human cell lines(Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88: 8972-8976). Inthis system, the gene of interest is subcloned into a vacciniarecombination plasmid such that the gene's open reading frame istranslationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni² +-nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

When used as a component in assay systems such as those described,below, in Section 5.5, the differentially expressed or pathway geneprotein may be labeled, either directly or indirectly, to facilitatedetection of a complex formed between the differentially expressed orpathway gene protein and a test substance Any of a variety of suitablelabeling systems may be used including but not limited to radioisotopessuch as ¹²⁵ I; enzyme labelling systems that generate a detectablecolorimetric signal or light when exposed to substrate; and fluorescentlabels.

Where recombinant DNA technology is used to produce the differentiallyexpressed or pathway gene protein for such assay systems, it may beadvantageous to engineer fusion proteins that can facilitate labeling,immobilization and/or detection.

Indirect labeling involves the use of a protein, such as a labeledantibody, which specifically binds to either a differentially expressedor pathway gene product Such antibodies include but are not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments andfragments produced by an Fab expression library.

5.4.3. Differentially Expressed or Pathway Gene Product Antibodies

Described herein are methods for the production of antibodies capable ofspecifically recognizing one or more differentially expressed or pathwaygene epitopes. Such antibodies may include, but are not limited topolyclonal antibodies, monoclonal antibodies (mAbs), humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. Such antibodies may be used, for example, in thedetection of a fingerprint, target, or pathway gene in a biologicalsample, or, alternatively, as a method for the inhibition of abnormaltarget gene activity. Thus, such antibodies may be utilized as part ofcardiovascular disease treatment methods, and/or may be used as part ofdiagnostic techniques whereby patients may be tested for abnormal levelsof fingerprint, target, or pathway gene proteins, or for the presence ofabnormal forms of the such proteins.

For the production of antibodies to a differentially expressed orpathway gene, various host animals may be immunized by injection with adifferentially expressed or pathway gene protein, or a portion thereof.Such host animals may include but are not limited to rabbits, mice, andrats, to name but a few. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

In a preferred embodiment, peptide sequences corresponding to aminosequences of target gene products were selected and submitted toResearch Genetics (Huntsville, Ala.) for synthesis and antibodyproduction. Peptides were modified as described (Tam, J. P., 1988, Proc.Natl. Acad. Sci. USA 85: 5409-5413; Tam, J. P., and Zavala, F., 1989, J.Immunol. Methods 124: 53-61; Tam, J. P., and Lu, Y. A., 1989, Proc.Natl. Acad. Sci. USA 86: 9084-9088), emulsified in an equal volume ofFreund's adjuvant and injected into rabbits at 3 to 4 subcutaneousdorsal sites for a total volume of 1.0 ml (0.5 mg peptide) perimmunization. The animals were boosted after 2 and 6 weeks and bled atweeks 4, 8, and 10. The blood was allowed to clot and serum wascollected by centrifugation. The generation of polyclonal antibodiesagainst the fchd545 gene product is described in detail in the examplein Section 10, below.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as target gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, may be immunized by injection withdifferentially expressed or pathway gene product supplemented withadjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture These include, but are not limited to the hybridoma technique ofKohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of "chimericantibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to producedifferentially expressed or pathway gene-single chain antibodies Singlechain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated byknown techniques For example, such fragments include but are not limitedto: the F(ab')₂ fragments which can be produced by pepsin digestion ofthe antibody molecule and the Fab fragments which can be generated byreducing the disulfide bridges of the F(ab')₂ fragments. Alternatively,Fab expression libraries may be constructed (Huse et al., 1989, Science,246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity.

5.4.4. Cell- and Animal-based Model Systems

Described herein are cell- and animal-based systems which act as modelsfor cardiovascular disease These systems may be used in a variety ofapplications. For example, the cell- and animal-based model systems maybe used to further characterize differentially expressed and pathwaygenes, as described, above, in Section 5.3. Such furthercharacterization may, for example, indicate that a differentiallyexpressed gene is a target gene. Second, such assays may be utilized aspart of screening strategies designed to identify compounds which arecapable of ameliorating cardiovascular disease symptoms, as described,below, in Section 5.5.4. Thus, the animal- and cell-based models may beused to identify drugs, pharmaceuticals, therapies and interventionswhich may be effective in treating cardiovascular disease. In addition,as described in detail, below, in Section 5.7.1, such animal models maybe used to determine the LD₅₀ and the ED₅₀ in animal subjects, and suchdata can be used to determine the in vivo efficacy of potentialcardiovascular disease treatments.

5.4.4.1. Animal-Based Systems

Animal-based model systems of cardiovascular disease may include, butare not limited to, non-recombinant and engineered transgenic animals.

Non-recombinant animal models for cardiovascular disease may include,for example, genetic models. Such genetic cardiovascular disease modelsmay include, for example, apob or apor deficient pigs (Rapacz, et al.,1986, Science 234:1573-1577) and Watanabe heritable hyperlipidemic(WHHL) rabbits (Kita et al., 1987, Proc. Natl. Acad. Sci USA 84:5928-5931).

Non-recombinant, non-genetic animal models of atherosclerosis mayinclude, for example, pig, rabbit, or rat models in which the animal hasbeen exposed to either chemical wounding through dietary supplementationof LDL, or mechanical wounding through balloon catheter angioplasty, forexample.

Additionally, animal models exhibiting cardiovascular disease symptomsmay be engineered by utilizing, for example, target gene sequences suchas those described, above, in Section 5.4.1, in conjunction withtechniques for producing transgenic animals that are well known to thoseof skill in the art. For example, target gene sequences may beintroduced into, and overexpressed in, the genome of the animal ofinterest, or, if endogenous target gene sequences are present, they mayeither be overexpressed or, alternatively, be disrupted in order tounderexpress or inactivate target gene expression, such as described forthe disruption of apoE in mice (Plump et al., 1992, Cell 71: 343-353).

In order to overexpress a target gene sequence, the coding portion ofthe target gene sequence may be ligated to a regulatory sequence whichis capable of driving gene expression in the animal and cell type ofinterest. Such regulatory regions will be well known to those of skillin the art, and may be utilized in the absence of undue experimentation.

For underexpression of an endogenous target gene sequence, such asequence may be isolated and engineered such that when reintroduced intothe genome of the animal of interest, the endogenous target gene alleleswill be inactivated. Preferably, the engineered target gene sequence isintroduced via gene targeting such that the endogenous target sequenceis disrupted upon integration of the engineered target gene sequenceinto the animal's genome. Gene targeting is discussed, below, in thisSection.

Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates,e.g., baboons, monkeys, and chimpanzees may be used to generatecardiovascular disease animal models.

Any technique known in the art may be used to introduce a target genetransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. Thetransgene may be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems. The transgene mayalso be selectively introduced into and activated in a particular celltype by following, for example, the teaching of Lasko et al. (Lasko, M.et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236). The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art. When it is desired that the target gene transgenebe integrated into the chromosomal site of the endogenous target gene,gene targeting is preferred. Briefly, when such a technique is to beutilized, vectors containing some nucleotide sequences homologous to theendogenous target gene of interest are designed for the purpose ofintegrating, via homologous recombination with chromosomal sequences,into and disrupting the function of the nucleotide sequence of theendogenous target gene. The transgene may also be selectively introducedinto a particular cell type, thus inactivating the endogenous gene ofinterest in only that cell type, by following, for example, the teachingof Gu et al. (Gu, et al., 1994, Science 265: 103-106). The regulatorysequences required for such a cell-type specific inactivation willdepend upon the particular cell type of interest, and will be apparentto those of skill in the art. Recombinant methods for expressing targetgenes are described in Section 5.4.2, above.

Once transgenic animals have been generated, the expression of therecombinant target gene and protein may be assayed utilizing standardtechniques. Initial screening may be accomplished by Southern blotanalysis or PCR techniques to analyze animal tissues to assay whetherintegration of the transgene has taken place. The level of mRNAexpression of the transgene in the tissues of the transgenic animals mayalso be assessed using techniques which include but are not limited toNorthern blot analysis of tissue samples obtained from the animal, insitu hybridization analysis, and RT-PCR. Samples of targetgene-expressing tissue, may also be evaluated immunocytochemically usingantibodies specific for the target gene transgene gene product ofinterest.

The target gene transgenic animals that express target gene mRNA ortarget gene transgene peptide (detected immunocytochemically, usingantibodies directed against the target gene product's epitopes) ateasily detectable levels should then be further evaluated to identifythose animals which display characteristic cardiovascular diseasesymptoms. Such symptoms may include, for example, increased prevalenceand size of fatty streaks and/or cardiovascular disease plaques.

Additionally, specific cell types within the transgenic animals may beanalyzed and assayed for cellular phenotypes characteristic ofcardiovascular disease. In the case of monocytes, such phenotypes mayinclude but are not limited to increases in rates of LDL uptake,adhesion to endothelial cells, transmigration, foam cell formation,fatty streak formation, and production of foam cell specific products.Cellular phenotype assays are discussed in detail in Section 5.4.4.2,below. Further, such cellular phenotypes may include a particular celltype's fingerprint pattern of expression as compared to knownfingerprint expression profiles of the particular cell type in animalsexhibiting cardiovascular disease symptoms. Fingerprint profiles aredescribed in detail in Section 5.8.1, below. Such transgenic animalsserve as suitable model systems for cardiovascular disease.

Once target gene transgenic founder animals are produced, they may bebred, inbred, outbred, or crossbred to produce colonies of theparticular animal. Examples of such breeding strategies include but arenot limited to: outbreeding of founder animals with more than oneintegration site in order to establish separate lines; inbreeding ofseparate lines in order to produce compound target gene transgenics thatexpress the target gene transgene of interest at higher levels becauseof the effects of additive expression of each target gene transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order both to augmentexpression and eliminate the possible need for screening of animals byDNA analysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; breeding animals to different inbredgenetic backgrounds so as to examine effects of modifying alleles onexpression of the target gene transgene and the development ofcardiovascular disease symptoms. One such approach is to cross thetarget gene transgenic founder animals with a wild type strain toproduce an F1 generation that exhibits cardiovascular disease symptoms.The F1 generation may then be inbred in order to develop a homozygousline, if it is found that homozygous target gene transgenic animals areviable

5.4.4.2. Cell-Based Assays

Cells that contain and express target gene sequences which encode targetgene protein, and, further, exhibit cellular phenotypes associated withcardiovascular disease, may be utilized to identify compounds thatexhibit anti-cardiovascular disease activity

Such cells may include non-recombinant monocyte cell lines, such as U937(ATCC# CRL-1593), THP-1 (ATCC# TIB-202), and P388D1 (ATCC# TIB-63);endothelial cells such as HUVEC's and bovine aortic endothelial cells(BAEC's); as well as generic mammalian cell lines such as HeLa cells andCOS cells, e.g., COS-7 (ATCC# CRL-1651). Further, such cells may includerecombinant, transgenic cell lines. For example, the cardiovasculardisease animal models of the invention, discussed, above, in Section5.4.4.1, may be used to generate cell lines, containing one or more celltypes involved in cardiovascular disease, that can be used as cellculture models for this disorder. While primary cultures derived fromthe cardiovascular disease transgenic animals of the invention may beutilized, the generation of continuous cell lines is preferred. Forexamples of techniques which may be used to derive a continuous cellline from the transgenic animals, see Small et al., 1985, Mol. CellBiol. 5:642-648.

Alternatively, cells of a cell type known to be involved incardiovascular disease may be transfected with sequences capable ofincreasing or decreasing the amount of target gene expression within thecell. For example, target gene sequences may be introduced into, andoverexpressed in, the genome of the cell of interest, or, if endogenoustarget gene sequences are present, they may be either overexpressed or,alternatively disrupted in order to underexpress or inactivate targetgene expression.

In order to overexpress a target gene sequence, the coding portion ofthe target gene sequence may be ligated to a regulatory sequence whichis capable of driving gene expression in the cell type of interest Suchregulatory regions will be well known to those of skill in the art, andmay be utilized in the absence of undue experimentation. Recombinantmethods for expressing target genes are described in Section 5.4.2,above.

For underexpression of an endogenous target gene sequence, such asequence may be isolated and engineered such that when reintroduced intothe genome of the cell type of interest, the endogenous target genealleles will be inactivated. Preferably, the engineered target genesequence is introduced via gene targeting such that the endogenoustarget sequence is disrupted upon integration of the engineered targetgene sequence into the cell's genome. Transfection of host cells withtarget genes is discussed, above, in Section 5.4.4.1.

Cells treated with compounds or transfected with target genes can beexamined for phenotypes associated with cardiovascular disease. In thecase of monocytes, such phenotypes include but are not limited toincreases in rates of LDL uptake, adhesion to endothelial cells,transmigration, foam cell formation, fatty streak formation, andproduction by foam cells of growth factors such as bFGF, IGF-I, VEGF,IL-1, M-CSF, TGFβ, TGFα, TNFα, HB-EGF, PDGF, IFN-γ, and GM-CSF.Transmigration rates, for example, may be measured using the in vitrosystem of Navab et al., described in Section 5.1.1.3, above, byquantifying the number of monocytes that migrate across the endothelialmonolayer and into the collagen layer of the subendothelial space.

Similarly, HUVEC's can be treated with test compounds or transfectedwith genetically engineered target genes described in Section 5.4.2,above. The HUVEC's can then be examined for phenotypes associated withcardiovascular disease, including, but not limited to changes incellular morphology, cell proliferation, cell migration, and mononuclearcell adhesion; or for the effects on production of other proteinsinvolved in cardiovascular disease such as ICAM, VCAM, PDGF-β, andE-selectin.

Transfection of target gene sequence nucleic acid may be accomplished byutilizing standard techniques. See, for example, Ausubel, 1989, supra.Transfected cells should be evaluated for the presence of therecombinant target gene sequences, for expression and accumulation oftarget gene mRNA, and for the presence of recombinant target geneprotein production. In instances wherein a decrease in target geneexpression is desired, standard techniques may be used to demonstratewhether a decrease in endogenous target gene expression and/or in targetgene product production is achieved.

5.5. Screening Assays for Compounds that Interact with the Target GeneProduct and/or Modulate Target Gene Expression

The following assays are designed to identify compounds that bind totarget gene products, bind to other cellular or extracellular proteinsthat interact with a target gene product, and interfere with theinteraction of the target gene product with other cellular orextracellular proteins. Such compounds can act as the basis foramelioration of such cardiovascular diseases as atherosclerosis,ischemia/reperfusion, hypertension, restenosis, and arterialinflammation by modulating the activity of the protein products oftarget genes. Such compounds may include, but are not limited topeptides, antibodies, or small organic or inorganic compounds. Methodsfor the identification of such compounds are described in Section 5.5.1,below. Such compounds may also include other cellular proteins. Methodsfor the identification of such cellular proteins are described, below,in Section 5.5.2.

Compounds identified via assays such as those described herein may beuseful, for example, in elaborating the biological function of thetarget gene product, and for ameliorating cardiovascular disease. Ininstances whereby a cardiovascular disease condition results from anoverall lower level of target gene expression and/or target gene productin a cell or tissue, compounds that interact with the target geneproduct may include compounds which accentuate or amplify the activityof the bound target gene protein. Such compounds would bring about aneffective increase in the level of target gene product activity, thusameliorating symptoms.

In some cases, a target gene observed to be up-regulated under diseaseconditions may be exerting a protective effect. Compounds that enhancethe expression of such up-regulated genes, or the activity of their geneproducts, would also ameliorate disease symptoms, especially inindividuals whose target gene is not normally up-regulated.

In other instances mutations within the target gene may cause aberranttypes or excessive amounts of target gene proteins to be made which havea deleterious effect that leads to cardiovascular disease. Similarly,physiological conditions may cause an excessive increase in target geneexpression leading to cardiovascular disease. In such cases, compoundsthat bind target gene protein may be identified that inhibit theactivity of the bound target gene protein Assays for testing theeffectiveness of compounds, identified by, for example, techniques suchas those described in this Section are discussed, below, in Section5.5.4.

5.5.1. In Vitro Screening Assays for Compounds that Bind to the TargetGene Product

In vitro systems may be designed to identify compounds capable ofbinding the target gene of the invention Such compounds may include, butare not limited to, peptides made of D-and/or L-configuration aminoacids (in, for example, the form of random peptide libraries; see e.g.,Lam, K. S. et al., 1991, Nature 354:82-84), phosphopeptides (in, forexample, the form of random or partially degenerate, directedphosphopeptide libraries; see, e.g., Songyang, Z. et al., 1993, Cell72:767-778), antibodies, and small organic or inorganic molecules.Compounds identified may be useful, for example, in modulating theactivity of target gene proteins, preferably mutant target geneproteins, may be useful in elaborating the biological function of thetarget gene protein, may be utilized in screens for identifyingcompounds that disrupt normal target gene interactions, or may inthemselves disrupt such interactions For instance, the example inSection 12, below, describes the interaction between the rchd534 proteinand the fchd540 protein. Compounds that disrupt the interaction betweenthese two proteins may be useful in the treatment of cardiovasculardisease.

The principle of the assays used to identify compounds that bind to thetarget gene protein involves preparing a reaction mixture of the targetgene protein and the test compound under conditions and for a timesufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoring thetarget gene or the test substance onto a solid phase and detectingtarget gene/test substance complexes anchored on the solid phase at theend of the reaction. In one embodiment of such a method, the target geneprotein may be anchored onto a solid surface, and the test compound,which is not anchored, may be labeled, either directly or indirectly.

In practice, microtitre plates are conveniently utilized. The anchoredcomponent may be immobilized by non-covalent or covalent attachments.Non-covalent attachment may be accomplished simply by coating the solidsurface with a solution of the protein and drying. Alternatively, animmobilized antibody, preferably a monoclonal antibody, specific for theprotein may be used to anchor the protein to the solid surface. Thesurfaces may be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously nonimmobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody)

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for target geneproduct or the test compound to anchor any complexes formed in solution,and a labeled antibody specific for the other component of the possiblecomplex to detect anchored complexes.

Compounds that are shown to bind to a particular target gene productthrough one of the methods described above can be further tested fortheir ability to elicit a biochemical response from the target geneprotein. A particular embodiment is described herein for receptorproteins involved in signal transduction. Compounds that interact with atarget gene product receptor domain, can be screened for their abilityto function as ligands, i.e., to bind to the receptor protein in amanner that triggers the signal transduction pathway. Useful receptorfragments or analogs in the invention are those which interact withligand. The receptor component can be assayed functionally, i.e., forits ability to bind ligand and mobilize Ca⁺⁺ (see below). These assaysinclude, as components, ligand and a recombinant target gene product (ora suitable fragment or analog) configured to permit detection ofbinding.

For example, and not by way of limitation, a recombinant receptor may beused to screen for ligands by its ability to mediate ligand-dependentmobilization of calcium. Cells, preferably myeloma cells or Xenopusoocytes, transfected with a target gene expression vector (constructedaccording to the methods described in Section 5.4.2, above) are loadedwith FURA-2 or INDO-1 by standard techniques.

Mobilization of Ca²⁺ induced by ligand is measured by fluorescencespectroscopy as previously described (Grynkiewicz et al., 1985, J. Biol.Chem. 260:3440). Ligands that react with the target gene productreceptor domain, therefore, can be identified by their ability toproduce a fluorescent signal. Their receptor binding activities can bequantified and compared by measuring the level of fluorescence producedover background. Identification of ligand, and measuring the activity ofthe ligand-receptor complex, leads to the identification of antagonistsof this interaction, as described in Section 5.5.3, below. Suchantagonists are useful in the treatment of cardiovascular disease.

5.5.2. Assays for Cellular or Extracellular Proteins that Interact withthe Target Gene Product

Any method suitable for detecting protein-protein interactions may beemployed for identifying novel target protein-cellular or extracellularprotein interactions. These methods are outlined in Section 5.2., supra,for the identification of pathway genes, and may be utilized herein withrespect to the identification of proteins which interact with identifiedtarget proteins. In such a case, the target gene serves as the known"bait" gene.

The example presented in Section 12, below, demonstrates the use of thismethod to detect the interaction between the rchd534 protein and thefchd540 protein, which both had been identified as target proteins.

5.5.3. Assays for Compounds that Interfere with Interaction betweenTarget Gene Product and Other Compounds

The target gene proteins of the invention may, in vivo, interact withone or more cellular or extracellular proteins. Such proteins mayinclude, but are not limited to, those proteins identified via methodssuch as those described, above, in Section 5.5.2. For the purposes ofthis discussion, target gene products and such cellular andextracellular proteins are referred to herein as "binding partners".Compounds that disrupt such interactions may be useful in regulating theactivity of the target gene proteins, especially mutant target geneproteins. Such compounds may include, but are not limited to moleculessuch as antibodies, peptides, and the like described in Section 5.5.1.above.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the target gene protein, and itscellular or extracellular protein binding partner or partners involvespreparing a reaction mixture containing the target gene protein and thebinding partner under conditions and for a time sufficient to allow thetwo proteins to interact and bind, thus forming a complex. In order totest a compound for inhibitory activity, the reaction mixture isprepared in the presence and absence of the test compound. The testcompound may be initially included in the reaction mixture or may beadded at a time subsequent to the addition of target gene and itscellular or extracellular binding partner. Control reaction mixtures areincubated without the test compound or with a placebo. The formation ofany complexes between the target gene protein and the cellular orextracellular binding partner is then detected. The formation of acomplex in the control reaction, but not in the reaction mixturecontaining the test compound, indicates that the compound interfereswith the interaction of the target gene protein and the interactivebinding partner protein. Additionally, complex formation within reactionmixtures containing the test compound and a normal target gene proteinmay also be compared to complex formation within reaction mixturescontaining the test compound and mutant target gene protein. Thiscomparison may be important in those cases wherein it is desirable toidentify compounds that disrupt interactions of mutant but not normaltarget gene proteins.

The assay for compounds that interfere with the interaction of thebinding partners can be conducted in a heterogeneous or homogeneousformat. Heterogeneous assays involve anchoring one of the bindingpartners onto a solid phase and detecting complexes anchored on thesolid phase at the end of the reaction. In homogeneous assays, theentire reaction is carried out in a liquid phase. In either approach,the order of addition of reactants can be varied to obtain differentinformation about the compounds being tested. For example, testcompounds that interfere with the interaction between the bindingpartners, e.g., by competition, can be identified by conducting thereaction in the presence of the test substance; i.e., by adding the testsubstance to the reaction mixture prior to or simultaneously with thetarget gene protein and interactive cellular or extracellular protein.Alternatively, test compounds that disrupt preformed complexes, e.g.compounds with higher binding constants that displace one of the bindingpartners from the complex, can be tested by adding the test compound tothe reaction mixture after complexes have been formed. The variousformats are described briefly below.

In a heterogeneous assay system, either the target gene protein or theinteractive cellular or extracellular binding partner protein, isanchored onto a solid surface, and its binding partner, which is notanchored, is labeled, either directly or indirectly. In practice,microtitre plates are conveniently utilized. The anchored species may beimmobilized by non-covalent or covalent attachments. Non-covalentattachment may be accomplished simply by coating the solid surface witha solution of the protein and drying. Alternatively, an immobilizedantibody specific for the protein may be used to anchor the protein tothe solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the binding partner of the immobilizedspecies is exposed to the coated surface with or without the testcompound. After the reaction is complete, unreacted components areremoved (e.g., by washing) and any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thebinding partner was pre-labeled, the detection of label immobilized onthe surface indicates that complexes were formed. Where the bindingpartner is not pre-labeled, an indirect label can be used to detectcomplexes anchored on the surface; e.g., using a labeled antibodyspecific for the binding partner (the antibody, in turn, may be directlylabeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one binding partner to anchor anycomplexes formed in solution, and a labeled antibody specific for theother binding partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the target gene proteinand the interactive cellular or extracellular protein is prepared inwhich one of the binding partners is labeled, but the signal generatedby the label is quenched due to complex formation (see, e.g., U.S. Pat.No. 4,109,496 by Rubenstein which utilizes this approach forimmunoassays). The addition of a test substance that competes with anddisplaces one of the binding partners from the preformed complex willresult in the generation of a signal above background. In this way, testsubstances which disrupt target gene protein-cellular or extracellularprotein interaction can be identified.

In a particular embodiment, the target gene protein can be prepared forimmobilization using recombinant DNA techniques described in Section5.4.2, supra. For example, the target gene coding region can be fused toa glutathione-S-transferase (GST) gene, using a fusion vector such asPGEX-5X-1, in such a manner that its binding activity is maintained inthe resulting fusion protein. The interactive cellular or extracellularprotein can be purified and used to raise a monoclonal antibody, usingmethods routinely practiced in the art and described above, in Section5.4.3. This antibody can be labeled with the radioactive isotope ¹²⁵ I,for example, by methods routinely practiced in the art. In aheterogeneous assay, e.g., the GST-target gene fusion protein can beanchored to glutathione-agarose beads The interactive cellular orextracellular binding partner protein can then be added in the presenceor absence of the test compound in a manner that allows interaction andbinding to occur. At the end of the reaction period, unbound materialcan be washed away, and the labeled monoclonal antibody can be added tothe system and allowed to bind to the complexed binding partners. Theinteraction between the target gene protein and the interactive cellularor extracellular binding partner protein can be detected by measuringthe amount of radioactivity that remains associated with theglutathione-agarose beads. A successful inhibition of the interaction bythe test compound will result in a decrease in measured radioactivity

Alternatively, the GST-target gene fusion protein and the interactivecellular or extracellular binding partner protein can be mixed togetherin liquid in the absence of the solid glutathione-agarose beads. Thetest compound can be added either during or after the binding partnersare allowed to interact. This mixture can then be added to theglutathione-agarose beads and unbound material is washed away. Again theextent of inhibition of the binding partner interaction can be detectedby adding the labeled antibody and measuring the radioactivityassociated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the target gene protein and the interactive cellular or extracellularprotein, respectively, in place of one or both of the full lengthproteins. Any number of methods routinely practiced in the art can beused to identify and isolate the protein's binding site. These methodsinclude, but are not limited to, mutagenesis of one of the genesencoding the proteins and screening for disruption of binding in aco-immunoprecipitation assay. Compensating mutations in the target genecan be selected Sequence analysis of the genes encoding the respectiveproteins will reveal the mutations that correspond to the region of theprotein involved in interactive binding. Alternatively, one protein canbe anchored to a solid surface using methods described in this Sectionabove, and allowed to interact with and bind to its labeled bindingpartner, which has been treated with a proteolytic enzyme, such astrypsin. After washing, a short, labeled peptide comprising the bindingdomain may remain associated with the solid material, which can beisolated and identified by amino acid sequencing. Also, once the genecoding for the for the cellular or extracellular protein is obtained,short gene segments can be engineered to express peptide fragments ofthe protein, which can then be tested for binding activity and purifiedor synthesized.

For example, and not by way of limitation, target gene can be anchoredto a solid material as described above in this Section by making aGST-target gene fusion protein and allowing it to bind to glutathioneagarose beads. The interactive cellular or extracellular binding partnerprotein can be labeled with a radioactive isotope, such as ³⁵ S, andcleaved with a proteolytic enzyme such as trypsin. Cleavage products canthen be added to the anchored GST-target gene fusion protein and allowedto bind. After washing away unbound peptides, labeled bound material,representing the cellular or extracellular binding partner proteinbinding domain, can be eluted, purified, and analyzed for amino acidsequence by techniques well known in the art; e.g., using the Edmandegradation procedure (see e.g., Creighton, 1983, Proteins: Structuresand Molecular Principles, W. H. Freeman & Co., N.Y., pp. 34-49).Peptides so identified can be produced, using techniques well known inthe art, either synthetically (see e.g., Creighton, 1983, supra at pp.50-60) or, if the gene has already been isolated, by using recombinantDNA technology, as described in Section 5.4.2, supra.

A particular embodiment of the invention features a method of screeningcandidate compounds for their ability to antagonize the interactionbetween ligand and the receptor domain of a target gene product. Themethod involves: a) mixing a candidate antagonist compound with a firstcompound which includes a recombinant target gene product comprising areceptor domain (or ligand-binding fragment or analog) on the one handand with a second compound which includes ligand on the other hand; b)determining whether the first and second compounds bind; and c)identifying antagonistic compounds as those which interfere with thebinding of the first compound to the second compound and/or which reducethe ligand-mediated release of intracellular Ca⁺⁺.

By an "antagonist" is meant a molecule which inhibits a particularactivity, in this case, the ability of ligand to interact with a targetgene product receptor domain and/or to trigger the biological eventsresulting from such an interaction (e.g., release of intracellularCa⁺⁺). Preferred therapeutics include antagonists, e.g., peptidefragments (particularly, fragments derived from the N-terminalextracellular domain), antibodies (particularly, antibodies whichrecognize and bind the N-terminal extracellular domain), or drugs, whichblock ligand or target gene product function by interfering with theligand-receptor interaction.

Because the receptor component of the target gene product can beproduced by recombinant techniques and because candidate antagonists maybe screened in vitro, the instant invention provides a simple and rapidapproach to the identification of useful therapeutics.

Specific receptor fragments of interest include any portions of thetarget gene products that are capable of interaction with ligand, forexample, all or part of the N-terminal extracellular domain. Suchportions include the transmembrane segments and portions of the receptordeduced to be extracellular. Such fragments may be useful as antagonists(as described above), and are also useful as immunogens for producingantibodies which neutralize the activity of the target gene product invivo (e.g., by interfering with the interaction between the receptor andligand; see below). Extracellular regions may be identified bycomparison with related proteins of similar structure, useful regionsare those exhibiting homology to the extracellular domains ofwell-characterized members of the family.

Alternatively, from the primary amino acid sequence, the secondaryprotein structure and, therefore, the extracellular domain regions maybe deduced semi-empirically using a hydrophobicity/hydrophilicitycalculation such as the Chou-Fasman method (see, e.g., Chou and Fasman,Ann. Rev. Biochem. 47:251, 1978). Hydrophilic domains, particularly onessurrounded by hydrophobic stretches (e.g., transmembrane domains)present themselves as strong candidates for extracellular domains.Finally, extracellular domains may be identified experimentally usingstandard enzymatic digest analysis, e.g., tryptic digest analysis.

Candidate fragments (e.g., all or part of the transmembrane segments orany extracellular fragment) are tested for interaction with ligand bythe assays described herein (e.g., the assay described above). Suchfragments are also tested for their ability to antagonize theinteraction between ligand and its endogenous receptor using the assaysdescribed herein. Analogs of useful receptor fragments (as describedabove) may also be produced and tested for efficacy as screeningcomponents or antagonists (using the assays described herein); suchanalogs are also considered to be useful in the invention.

Of particular interest are receptor fragments encompassing theextracellular main-terminal domain (or a ligand binding fragmentthereof). Also of interest are the target gene product extracellularloops. Peptide fragments derived from these extracellular loops may alsobe used as antagonists, particularly if the loops cooperate with theamino-terminal domain to facilitate ligand binding. Alternatively, suchloops and extracellular N-terminal domain (as well as the full lengthtarget gene product) provide immunogens for producing anti-target geneproduct antibodies.

Binding of ligand to its receptor may be assayed by any of the methodsdescribed above in Section 5.5.1. Preferably, cells expressingrecombinant target gene product (or a suitable target gene productfragment or analog) are immobilized on a solid substrate (e.g., the wallof a microtitre plate or a column) and reacted with detectably-labelledligand (as described above). Binding is assayed by the detection labelin association with the receptor component (and, therefore, inassociation with the solid substrate). Binding of labelled ligand toreceptor-bearing cells is used as a "control" against which antagonistassays are measured. The antagonist assays involve incubation of thetarget gene product-bearing cells with an appropriate amount ofcandidate antagonist. To this mix, an equivalent amount to labelledligand is added. An antagonist useful in the invention specificallyinterferes with labelled ligand binding to the immobilizedreceptor-expressing cells.

An antagonist is then tested for its ability to interfere with ligandfunction, i.e., to specifically interfere with labelled ligand bindingwithout resulting in signal transduction normally mediated by thereceptor. To test this using a functional assay, stably transfected celllines containing the target gene product can be produced as describedherein and reporter compounds such as the calcium binding agent, FURA-2,loaded into the cytoplasm by standard techniques. Stimulation of theheterologous target gene product with ligand or another agonist leads tointracellular calcium release and the concomitant fluorescence of thecalcium-FURA-2 complex. This provides a convenient means for measuringagonist activity. Inclusion of potential antagonists along with ligandallows for the screening and identification of authentic receptorantagonists as those which effectively block ligand binding withoutproducing fluorescence (i.e., without causing the mobilization ofintracellular Ca⁺⁺) Such an antagonist may be expected to be a usefultherapeutic agent for cardiovascular disorders

Appropriate candidate antagonists include target gene product fragments,particularly fragments containing a ligand-binding portion adjacent toor including one or more transmembrane segments or an extracellulardomain of the receptor (described above); such fragments wouldpreferably including five or more amino acids. Other candidateantagonists include analogs of ligand and other peptides as well asnon-peptide compounds and anti-target gene product antibodies designedor derived from analysis of the receptor

5.5.4. Assays for Amelioration of Cardiovascular Disease Symptons

Any of the binding compounds, including but not limited to compoundssuch as those identified in the foregoing assay systems, may be testedfor the ability to ameliorate cardiovascular disease symptoms.Cell-based and animal model-based assays for the identification ofcompounds exhibiting such an ability to ameliorate cardiovasculardisease symptoms are described below.

First, cell-based systems such as those described, above, in Section5.4.4.2., may be used to identify compounds which may act to amelioratecardiovascular disease symptoms For example, such cell systems may beexposed to a compound, suspected of exhibiting an ability to amelioratecardiovascular disease symptoms, at a sufficient concentration and for atime sufficient to elicit such an amelioration of cardiovascular diseasesymptoms in the exposed cells. After exposure, the cells are examined todetermine whether one or more of the cardiovascular disease cellularphenotypes has been altered to resemble a more normal or more wild type,non-cardiovascular disease phenotype. For example, and not by way oflimitation, in the case of monocytes, such more normal phenotypes mayinclude but are not limited to decreased rates of LDL uptake, adhesionto endothelial cells, transmigration, foam cell formation, fatty streakformation, and production by foam cells of growth factors such as bFGF,IGF-I, VEGF, IL-1, M-CSF, TGFβ, TGFα, TNFα, HB-EGF, PDGF, IFN-γ, andGM-CSF. Transmigration rates, for example, may be measured using the invitro system of Navab et al., described in Section 5.1.1.3, above, byquantifying the number of monocytes that migrate across the endothelialmonolayer and into the collagen layer of the subendothelial space

In addition, animal-based cardiovascular disease systems, such as thosedescribed, above, in Section 5.4.4.1, may be used to identify compoundscapable of ameliorating cardiovascular disease symptoms. Such animalmodels may be used as test substrates for the identification of drugs,pharmaceuticals, therapies, and interventions which may be effective intreating cardiovascular disease. For example, animal models may beexposed to a compound, suspected of exhibiting an ability to amelioratecardiovascular disease symptoms, at a sufficient concentration and for atime sufficient to elicit such an amelioration of cardiovascular diseasesymptoms in the exposed animals. The response of the animals to theexposure may be monitored by assessing the reversal of disordersassociated with cardiovascular disease, for example, by counting thenumber of atherosclerotic plaques and/or measuring their size before andafter treatment.

With regard to intervention, any treatments which reverse any aspect ofcardiovascular disease symptoms should be considered as candidates forhuman cardiovascular disease therapeutic intervention. Dosages of testagents may be determined by deriving dose-response curves, as discussedin Section 5.7.1, below.

Additionally, gene expression patterns may be utilized to assess theability of a compound to ameliorate cardiovascular disease symptoms. Forexample, the expression pattern of one or more fingerprint genes mayform part of a "fingerprint profile" which may be then be used in suchan assessment. "Fingerprint profile", as used herein, refers to thepattern of mRNA expression obtained for a given tissue or cell typeunder a given set of conditions. Such conditions may include, but arenot limited to, atherosclerosis, ischemia/reperfusion, hypertension,restenosis, and arterial inflammation, including any of the control orexperimental conditions described in the paradigms of Section 5.1.1,above. Fingerprint profiles may be generated, for example, by utilizinga differential display procedure, as discussed, above, in Section 5.1.2,Northern analysis and/or RT-PCR. Any of the gene sequences described,above, in Section 5.4.1 may be used as probes and/or PCR primers for thegeneration and corroboration of such fingerprint profiles.

Fingerprint profiles may be characterized for known states, eithercardiovascular disease or normal, within the cell- and/or animal-basedmodel systems. Subsequently, these known fingerprint profiles may becompared to ascertain the effect a test compound has to modify suchfingerprint profiles, and to cause the profile to more closely resemblethat of a more desirable fingerprint

For example, administration of a compound may cause the fingerprintprofile of a cardiovascular disease model system to more closelyresemble the control system Administration of a compound may,alternatively, cause the fingerprint profile of a control system tobegin to mimic a cardiovascular disease state. Such a compound may, forexample, be used in further characterizing the compound of interest, ormay be used in the generation of additional animal models.

5.5.5. Monitoring of Effects during Clinical Trials

Monitoring the influence of compounds on cardiovascular disease statesmay be applied not only in basic drug screening, but also in clinicaltrials. In such clinical trials, the expression of a panel of genes thathave been discovered in one of the paradigms described in Section5.1.1.1 through 5.1.1.6 may be used as a "read out" of a particulardrug's effect on a cardiovascular disease state.

For example, and not by way of limitation, Paradigm A provides for theidentification of fingerprint genes that are up-regulated in monocytestreated with oxidized LDL. Thus, to study the effect of anti-oxidantdrugs, for example, in a clinical trial, blood may be drawn frompatients before and at different stages during treatment with such adrug. Their monocytes may then be isolated and RNA prepared and analyzedby differential display as described in Sections 6.1.1 and 6.1.2. Thelevels of expression of these fingerprint genes may be quantified byNorthern blot analysis or RT-PCR, as described in Section 6.1.2, or byone of the methods described in Section 5.8.1, or alternatively bymeasuring the amount of protein produced, by one of the methodsdescribed in Section 5.8.2. In this way, the fingerprint profiles mayserve as surrogate markers indicative of the physiological response ofmonocytes that have taken up oxidized LDL. Accordingly, this responsestate may be determined before, and at various points during, drugtreatment. This method is described in further detail in the example inSection 8, below. Specifically, the up-regulation of fchd602 and fchd605under treatment with oxidized LDL provides a fingerprint profile formonocytes under oxidative stress. The fchd602 and fchd605 genes canserve, therefore, as surrogate markers during clinical treatment ofcardiovascular disease. Accordingly, the influence of anti-oxidant drugson oxidative potential is measured by recording the differential displayof fchd602 and fchd605 in the monocytes of patients undergoing clinicaltreatment

5.5.6. Assays for Compounds that Modulate Expression of Target Genes

Compounds and other substances that modulate expression of target genescan be screened using in vitro cellular systems. In a manner analogousto the monitoring of compounds clinical samples described in Section5.5.5, above, a sample of cells, such as a tissue culture is exposed toa test substance. Appropriate tissue culture cells include, but are notlimited to, human umbilical vein endothelial cells (HUVECs), bovineaortic endothelial cells (BAECs), and 293 cells (embryonic human kidneycells). The RNA is then extracted from the cells. The level oftranscription of a specific target gene can be detected using, forexample, standard RT-PCR amplification techniques and/or Northernanalysis (as described in the example in Section 6.1.2, below).Alternatively, the level of target protein production can be assayed byusing antibodies that detect the target gene protein, as described inSection 5.5.1, above The level of expression is compared to a controlcell sample which was not exposed to the test substance

Compounds that can be screened for modulation of expression of thetarget gene include, but are not limited to, small inorganic or organicmolecules, peptides, such as peptide hormones analogs, steroid hormones,analogs of such hormones, and other proteins. Compounds thatdown-regulate expression include, but are not limited to,oligonucleotides that are complementary to the 5'-end of the mRNA of thetarget gene and inhibit transcription by forming triple helixstructures, and ribozymes or antisense molecules which inhibittranslation of the target gene mRNA. Techniques and strategies fordesigning such down-regulating test compounds are described in detail inSection 5.6, below.

5.6. Compounds and Methods for Treatment of Cardiovascular Disease

Described below are methods and compositions whereby cardiovasculardisease symptoms may be ameliorated. Certain cardiovascular diseases arebrought about, at least in part, by an excessive level of gene product,or by the presence of a gene product exhibiting an abnormal or excessiveactivity. As such, the reduction in the level and/or activity of suchgene products would bring about the amelioration of cardiovasculardisease symptoms. Techniques for the reduction of target gene expressionlevels or target gene product activity levels are discussed in Section5.6.1, below.

Alternatively, certain other cardiovascular diseases are brought about,at least in part, by the absence or reduction of the level of geneexpression, or a reduction in the level of a gene product's activity. Assuch, an increase in the level of gene expression and/or the activity ofsuch gene products would bring about the amelioration of cardiovasculardisease symptoms.

In some cases, the up-regulation of a gene in a disease state reflects aprotective role for that gene product in responding to the diseasecondition. Enhancement of such a target gene's expression, or theactivity of the target gene product, will reinforce the protectiveeffect it exerts. Some cardiovascular disease states may result from anabnormally low level of activity of such a protective gene. In thesecases also, an increase in the level of gene expression and/or theactivity of such gene products would bring about the amelioration ofcardiovascular disease symptoms. Techniques for increasing target geneexpression levels or target gene product activity levels are discussedin Section 5.6.2, below.

5.6.1. Compounds that Inhibit Expression, Synthesis or Activity ofMutant Target Gene Activity

As discussed above, target genes involved in cardiovascular diseasedisorders can cause such disorders via an increased level of target geneactivity. As summarized in Table 1, above, and detailed in the examplesin Sections 6 and 7, below, a number of genes have been demonstrated tobe up-regulated in monocytes and endothelial cells under diseaseconditions Specifically, fchd602 and fchd605 are each up-regulated inmonocytes treated with oxidized LDL. Furthermore, fchd540 isup-regulated in endothelial cells subjected to shear stress. In somecases, such up-regulation may have a causative or exacerbating effect onthe disease state. A variety of techniques may be utilized to inhibitthe expression, synthesis, or activity of such target genes and/orproteins.

For example, compounds such as those identified through assaysdescribed, above, in Section 5.5, which exhibit inhibitory activity, maybe used in accordance with the invention to ameliorate cardiovasculardisease symptoms. As discussed in Section 5.5, above, such molecules mayinclude, but are not limited to small organic molecules, peptides,antibodies, and the like. Inhibitory antibody techniques are described,below, in Section 5.6.1.2.

For example, compounds can be administered that compete with endogenousligand for a transmembrane target gene product. The resulting reductionin the amount of ligand-bound target gene transmembrane protein willmodulate cell physiology. Compounds that can be particularly useful forthis purpose include, for example, soluble proteins or peptides, such aspeptides comprising one or more of the extracellular domains, orportions and/or analogs thereof, of the target gene product, including,for example, soluble fusion proteins such as Ig-tailed fusion proteins.(For a discussion of the production of Ig-tailed fusion proteins, see,for example, U.S. Pat. No. 5,116,964.). Alternatively, compounds, suchas ligand analogs or antibodies, that bind to the target gene productreceptor site, but do not activate the protein, (e.g., receptor-ligandantagonists) can be effective in inhibiting target gene productactivity.

Further, antisense and ribozyme molecules which inhibit expression ofthe target gene may also be used in accordance with the invention toinhibit the aberrant target gene activity. Such techniques aredescribed, below, in Section 5.6.1.1. Still further, also as described,below, in Section 5.6.1.1, triple helix molecules may be utilized ininhibiting the aberrant target gene activity.

5.6.1.1. Inhibitory Antisense, Ribozyme, Triple Helix, and GeneInactivation Approaches

Among the compounds which may exhibit the ability to amelioratecardiovascular disease symptoms are antisense, ribozyme, and triplehelix molecules. Such molecules may be designed to reduce or inhibitmutant target gene activity Techniques for the production and use ofsuch molecules are well known to those of skill in the art.

Antisense RNA and DNA molecules act to directly block the translation ofmRNA by hybridizing to targeted mRNA and preventing protein translation.

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to target gene mRNA. The antisenseoligonucleotides will bind to the complementary target gene mRNAtranscripts and prevent translation Absolute complementarity, althoughpreferred, is not required A sequence "complementary" to a portion of anRNA, as referred to herein, means a sequence having sufficientcomplementarity to be able to hybridize with the RNA, forming a stableduplex; in the case of double-stranded antisense nucleic acids, a singlestrand of the duplex DNA may thus be tested, or triplex formation may beassayed The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense nucleic acid. Generally,the longer the hybridizing nucleic acid, the more base mismatches withan RNA it may contain and still form a stable duplex (or triplex, as thecase may be). One skilled in the art can ascertain a tolerable degree ofmismatch by use of standard procedures to determine the melting point ofthe hybridized complex

Oligonucleotides that are complementary to the 5' end of the message,e.g., the 5' untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibiting translationHowever, sequences complementary to the 3' untranslated sequences ofmRNAs have recently shown to be effective at inhibiting translation ofmRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-335.Thus, oligonucleotides complementary to either the 5'- or3'-non-translated, non-coding regions of the target gene could be usedin an antisense approach to inhibit translation of endogenous targetgene mRNA. Oligonucleotides complementary to the 5' untranslated regionof the mRNA should include the complement of the AUG start codon.Antisense oligonucleotides complementary to mRNA coding regions are lessefficient inhibitors of translation but could be used in accordance withthe invention. Whether designed to hybridize to the 5'-, 3'- or codingregion of target gene mRNA, antisense nucleic acids should be at leastsix nucleotides in length, and are preferably oligonucleotides rangingfrom 6 to about 50 nucleotides in length. In specific aspects theoligonucleotide is at least 10 nucleotides, at least 17 nucleotides, atleast 25 nucleotides or at least 50 nucleotides.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the target RNAor protein with that of an internal control RNA or protein.Additionally, it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo) or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556, Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5'-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group consistingof a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2'-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

While antisense nucleotides complementary to the target gene codingregion sequence could be used, those complementary to the transcribeduntranslated region are most preferred.

Specific antisense oligonucleotides for the rchd534 gene and fchd540gene are described in the Example in Section 13, below.

The antisense molecules should be delivered to cells which express thetarget gene in vivo, e.g., endothelial cells. A number of methods havebeen developed for delivering antisense DNA or RNA to cells; e.g.,antisense molecules can be injected directly into the tissue site, ormodified antisense molecules, designed to target the desired cells(e.g., antisense linked to peptides or antibodies that specifically bindreceptors or antigens expressed on the target cell surface) can beadministered systemically

However, it is often difficult to achieve intracellular concentrationsof the antisense sufficient to suppress translation of endogenous mRNAs.Therefore a preferred approach utilizes a recombinant DNA construct inwhich the antisense oligonucleotide is placed under the control of astrong pol III or pol II promoter. The use of such a construct totransfect target cells in the patient will result in the transcriptionof sufficient amounts of single stranded RNAs that will formcomplementary base pairs with the endogenous target gene transcripts andthereby prevent translation of the target gene mRNA. For example, avector can be introduced in vivo such that it is taken up by a cell anddirects the transcription of an antisense RNA. Such a vector can remainepisomal or become chromosomally integrated, as long as it can betranscribed to produce the desired antisense RNA. Such vectors can beconstructed by recombinant DNA technology methods standard in the art.Vectors can be plasmid, viral, or others known in the art, used forreplication and expression in mammalian cells. Expression of thesequence encoding the antisense RNA can be by any promoter known in theart to act in mammalian, preferably human cells. Such promoters can beinducible or constitutive. Such promoters include but are not limitedto: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3' long terminal repeat ofRous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:39-42), etc. Any type ofplasmid, cosmid, YAC or viral vector can be used to prepare therecombinant DNA construct which can be introduced directly into thetissue site; e.g., atherosclerotic vascular tissue. Alternatively, viralvectors can be used which selectively infect the desired tissue, inwhich case administration may be accomplished by another route (e.g.,systemically).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involves sequencespecific hybridization of the ribozyme molecule to complementary targetRNA, followed by an endonucleolytic cleavage. Ribozyme moleculesdesigned to catalytically cleave target gene mRNA transcripts can alsobe used to prevent translation of target gene mRNA and expression oftarget gene. (See, e.g., PCT International Publication WO90/11364,published Oct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225).While ribozymes that cleave mRNA at site specific recognition sequencescan be used to destroy target gene mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5'-UG-3'. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591.For example, there are hundreds of potential hammerhead ribozymecleavage sites within the nucleotide sequence of rchd534 and fchd540cDNA. Preferably the ribozyme is engineered so that the cleavagerecognition site is located near the 5' end of the target mRNA; i.e., toincrease efficiency and minimize the intracellular accumulation ofnon-functional mRNA transcripts

Specific hammerhead ribozymes molecules for the rchd534 and fchd540genes are described in the Example in Section 13, below.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter "Cech-type ribozymes") such as the onewhich occurs naturally in Tetrahymena Thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429-433; published International patent application No. WO 88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes which targeteight base-pair active site sequences that are present in target gene.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g. for improved stability, targeting, etc.) andshould be delivered to cells which express the target gene in vivo,e.g., endothelial cells. A preferred method of delivery involves using aDNA construct "encoding" the ribozyme under the control of a strongconstitutive pol III or pol II promoter, so that transfected cells willproduce sufficient quantities of the ribozyme to destroy endogenoustarget gene messages and inhibit translation. Because ribozymes, unlikeantisense molecules, are catalytic, a lower intracellular concentrationis required for efficiency.

Nucleic acid molecules to be used in triple helix formation for theinhibition of transcription should be single stranded and composed ofdeoxyribonucleotides. The base composition of these oligonucleotidesmust be designed to promote triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of eitherpurines or pyrimidines to be present on one strand of a duplex.Nucleotide sequences may be pyrimidine-based, which will result in TATand CGC⁺ triplets across the three associated strands of the resultingtriple helix. The pyrimidine-rich molecules provide base complementarityto a purine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC paris, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in GGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called "switchback"nucleic acid molecule. Switchback molecules are synthesized in analternating 5'-3', 3'-5' manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizeable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

It is possible that the antisense, ribozyme, and/or triple helixmolecules described herein may reduce or inhibit the transcription(triple helix) and/or translation (antisense, ribozyme) of mRNA producedby both normal and mutant target gene alleles. In order to ensure thatsubstantially normal levels of target gene activity are maintained,nucleic acid molecules that encode and express target gene polypeptidesexhibiting normal activity may be introduced into cells via gene therapymethods such as those described, below, in Section 5.7. that do notcontain sequences susceptible to whatever antisense, ribozyme, or triplehelix treatments are being utilized. Alternatively, it may be preferableto coadminister normal target gene protein into the cell or tissue inorder to maintain the requisite level of cellular or tissue target geneactivity.

Endogenous target gene expression can also be reduced by inactivating or"knocking out" the target gene or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., 1985, Nature 317:230-234;Thomas & Capecchi, 1987, Cell 51:503-512; Thompson et al., 1989 Cell5:313-321; each of which is incorporated by reference herein in itsentirety). For example, a mutant, non-functional target (or a completelyunrelated DNA sequence) flanked by DNA homologous to the endogenoustarget gene (either the coding regions or regulatory regions of thetarget gene) can be used, with or without a selectable marker and/or anegative selectable marker, to transfect cells that express target invivo. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the target gene. Suchapproaches can be adapted for use in humans provided the recombinant DNAconstructs are directly administered or targeted to the required site invivo using appropriate viral vectors, e.g., vectors for deliveryvascular tissue.

Alternatively, endogenous target gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the target gene (i.e., the target promoter and/or enhancers)to form triple helical structures that prevent transcription of thetarget gene in target cells in the body. (See generally, Helene, C.1991, Anticancer Drug Des., 6(6):569-84; Helene, C., et al., 1992, Ann,N.Y. Acad. Sci., 660:27-36; and Maher, L. J., 1992, Bioassays14(12):807-15).

In yet another embodiment of the invention, the activity of a target canbe reduced using a "dominant negative" approach to effectuate reductionin cardiovascular disease symptoms. For example, if two gene productsinteract, such as the rchd534 and fchd540 proteins, then the presence ofa mutant version of one or both of these proteins in the cell can reducethe overall pool of complexes consisting of entirely wild-type proteins.In this manner, the overall level of activity resulting from therchd534/fchd540 protein interaction can be reduced.

5.6.1.2. Antibodies for Target Gene Products

Antibodies that are both specific for target gene protein and interferewith its activity may be used to inhibit target gene function. Suchantibodies may be generated using standard techniques described inSection 5.4.3., supra, against the proteins themselves or againstpeptides corresponding to portions of the proteins. Such antibodiesinclude but are not limited to polyclonal, monoclonal, Fab fragments,single chain antibodies, chimeric antibodies, etc.

In instances where the target gene protein is intracellular and wholeantibodies are used, internalizing antibodies may be preferred. However,lipofectin liposomes may be used to deliver the antibody or a fragmentof the Fab region which binds to the target gene epitope into cells.Where fragments of the antibody are used, the smallest inhibitoryfragment which binds to the target protein's binding domain ispreferred. For example, peptides having an amino acid sequencecorresponding to the domain of the variable region of the antibody thatbinds to the target gene protein may be used. Such peptides may besynthesized chemically or produced via recombinant DNA technology usingmethods well known in the art (e.g., see Creighton, 1983, supra; andSambrook et al., 1989, supra). Alternatively, single chain neutralizingantibodies which bind to intracellular target gene epitopes may also beadministered. Such single chain antibodies may be administered, forexample, by expressing nucleotide sequences encoding single-chainantibodies within the target cell population by utilizing, for example,techniques such as those described in Marasco et al. (Marasco, W. etal., 1993, Proc. Natl. Acad. Sci. USA 90:7889-7893).

In some instances, the target gene protein is extracellular, or is atransmembrane protein, such as the fchd545 and fchd602 gene products.Antibodies that are specific for one or more extracellular domains ofthese gene products, for example, and that interfere with its activity,are particularly useful in treating cardiovascular disease. Suchantibodies are especially efficient because they can access the targetdomains directly from the bloodstream Any of the administrationtechniques described, below in Section 5.7 which are appropriate forpeptide administration may be utilized to effectively administerinhibitory target gene antibodies to their site of action.

5.6.2. Methods for Restoring or Enhancing Target Gene Activity

Target genes that cause cardiovascular disease may be underexpressedwithin cardiovascular disease situations As summarized in Table 1,above, and detailed in the example in Section 7, below, several genesare now known to be down-regulated in endothelial cells under diseaseconditions. Specifically, fchd531 and fchd545 are down-regulated inendothelial cells subjected to shear stress. Alternatively, the activityof target gene products may be decreased, leading to the development ofcardiovascular disease symptoms. Such down-regulation of target geneexpression or decrease of target gene product activity might have acausative or exacerbating effect on the disease state.

In some cases, target genes that are up-regulated in the disease statemight be exerting a protective effect. As summarized in Table 1, above,and detailed in the examples in Sections 6 and 7, below, a number ofgenes are now known to be up-regulated in monocytes and endothelialcells under disease conditions. Specifically, fchd602 and fchd605 areeach up-regulated in monocytes treated with oxidized LDL. Furthermore,fchd540 is up-regulated in endothelial cells subjected to shear stress.A variety of techniques may be utilized to increase the expression,synthesis, or activity of such target genes and/or proteins, for thosegenes that exert a protective effect in response to disease conditions.

Described in this Section are methods whereby the level of target geneactivity may be increased to levels wherein cardiovascular diseasesymptoms are ameliorated. The level of gene activity may be increased,for example, by either increasing the level of target gene productpresent or by increasing the level of active target gene product whichis present.

For example, a target gene protein, at a level sufficient to amelioratecardiovascular disease symptoms may be administered to a patientexhibiting such symptoms. Any of the techniques discussed, below, inSection 5.7, may be utilized for such administration. One of skill inthe art will readily know how to determine the concentration ofeffective, non-toxic doses of the normal target gene protein, utilizingtechniques such as those described, below, in Section 5.7.1.

Additionally, RNA sequences encoding target gene protein may be directlyadministered to a patient exhibiting cardiovascular disease symptoms, ata concentration sufficient to produce a level of target gene proteinsuch that cardiovascular disease symptoms are ameliorated. Any of thetechniques discussed, below, in Section 5.7, which achieve intracellularadministration of compounds, such as, for example, liposomeadministration, may be utilized for the administration of such RNAmolecules. The RNA molecules may be produced, for example, byrecombinant techniques such as those described, above, in Section 5.4.2.

Further, patients may be treated by gene replacement therapy. One ormore copies of a normal target gene, or a portion of the gene thatdirects the production of a normal target gene protein with target genefunction, may be inserted into cells using vectors which include, butare not limited to adenovirus, adeno-associated virus, and retrovirusvectors, in addition to other particles that introduce DNA into cells,such as liposomes. Additionally, techniques such as those describedabove may be utilized for the introduction of normal target genesequences into human cells.

Cells, preferably, autologous cells, containing normal target geneexpressing gene sequences may then be introduced or reintroduced intothe patient at positions which allow for the amelioration ofcardiovascular disease symptoms. Such cell replacement techniques may bepreferred, for example, when the target gene product is a secreted,extracellular gene product.

5.7. Pharmaceutical Preparations and Methods of Administration

The identified compounds that inhibit target gene expression, synthesisand/or activity can be administered to a patient at therapeuticallyeffective doses to treat or ameliorate cardiovascular disease. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms of cardiovasculardisease.

5.7.1. Effective Dose

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀ /ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

5.7.2. Formulations and Use

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration

5.8. Diagnosis of Cardiovascular Disease Abnormalities

A variety of methods may be employed, utilizing reagents such asfingerprint gene nucleotide sequences described in Section 5.4.1, andantibodies directed against differentially expressed and pathway genepeptides, as described, above, in Sections 5.4.2. (peptides) and 5.4.3.(antibodies). Specifically, such reagents may be used, for example, forthe detection of the presence of target gene mutations, or the detectionof either over or under expression of target gene mRNA.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one specificfingerprint gene nucleic acid or anti-fingerprint gene antibody reagentdescribed herein, which may be conveniently used, e.g., in clinicalsettings, to diagnose patients exhibiting cardiovascular diseasesymptoms or at risk for developing cardiovascular disease.

Any cell type or tissue, preferably monocytes, endothelial cells, orsmooth muscle cells, in which the fingerprint gene is expressed may beutilized in the diagnostics described below.

5.8.1. Detection of Fingerprint Gene Nucleic Acids

DNA or RNA from the cell type or tissue to be analyzed may easily beisolated using procedures which are well known to those in the art.Diagnostic procedures may also be performed "in situ" directly upontissue sections (fixed and/or frozen) of patient tissue obtained frombiopsies or resections, such that no nucleic acid purification isnecessary. Nucleic acid reagents such as those described in Section 5.1.may be used as probes and/or primers for such in situ procedures (see,for example, Nuovo, G. J., 1992, PCR in situ hybridization: protocolsand applications, Raven Press, NY).

Fingerprint gene nucleotide sequences, either RNA or DNA, may, forexample, be used in hybridization or amplification assays of biologicalsamples to detect cardiovascular disease-related gene structures andexpression. Such assays may include, but are not limited to, Southern orNorthern analyses, single stranded conformational polymorphism analyses,in situ hybridization assays, and polymerase chain reaction analyses.Such analyses may reveal both quantitative aspects of the expressionpattern of the fingerprint gene, and qualitative aspects of thefingerprint gene expression and/or gene composition. That is, suchaspects may include, for example, point mutations, insertions,deletions, chromosomal rearrangements, and/or activation or inactivationof gene expression.

Preferred diagnostic methods for the detection of fingerprintgene-specific nucleic acid molecules may involve for example, contactingand incubating nucleic acids, derived from the cell type or tissue beinganalyzed, with one or more labeled nucleic acid reagents as aredescribed in Section 5.1, under conditions favorable for the specificannealing of these reagents to their complementary sequences within thenucleic acid molecule of interest. Preferably, the lengths of thesenucleic acid reagents are at least 9 to 30 nucleotides. Afterincubation, all non-annealed nucleic acids are removed from the nucleicacid fingerprint molecule hybrid. The presence of nucleic acids from thefingerprint tissue which have hybridized, if any such molecules exist,is then detected. Using such a detection scheme, the nucleic acid fromthe tissue or cell type of interest may be immobilized, for example, toa solid support such as a membrane, or a plastic surface such as that ona microtitre plate or polystyrene beads. In this case, after incubation,non-annealed, labeled fingerprint nucleic acid reagents of the typedescribed in Section 5.1. are easily removed. Detection of theremaining, annealed, labeled nucleic acid reagents is accomplished usingstandard techniques well-known to those in the art.

Alternative diagnostic methods for the detection of fingerprint genespecific nucleic acid molecules may involve their amplification, e.g.,by PCR (the experimental embodiment set forth in Mullis, K. B., 1987,U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, F., 1991, Proc.Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication(Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878),transcriptional amplification system (Kwoh, D. Y et al., 1989, Proc.Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. etal., 1988, Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In one embodiment of such a detection scheme, a cDNA molecule isobtained from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). Cell types or tissues fromwhich such RNA may be isolated include any tissue in which wild typefingerprint gene is known to be expressed, including, but not limited,to monocytes, endothelium, and/or smooth muscle. A fingerprint sequencewithin the cDNA is then used as the template for a nucleic acidamplification reaction, such as a PCR amplification reaction, or thelike. The nucleic acid reagents used as synthesis initiation reagents(e.g., primers) in the reverse transcription and nucleic acidamplification steps of this method are chosen from among the fingerprintgene nucleic acid reagents described in Section 5.1. The preferredlengths of such nucleic acid reagents are at least 15-30 nucleotides.For detection of the amplified product, the nucleic acid amplificationmay be performed using radioactively or non-radioactively labelednucleotides. Alternatively, enough amplified product may be made suchthat the product may be visualized by standard ethidium bromide stainingor by utilizing any other suitable nucleic acid staining method.

In addition to methods which focus primarily on the detection of onenucleic acid sequence, fingerprint profiles, as discussed in Section5.5.4, may also be assessed in such detection schemes. Fingerprintprofiles may be generated, for example, by utilizing a differentialdisplay procedure, as discussed, above, in Section 5.1.2, Northernanalysis and/or RT-PCR. Any of the gene sequences described, above, inSection 5.4.1. may be used as probes and/or PCR primers for thegeneration and corroboration of such fingerprint profiles.

5.8.2. Detection of Fingerprint Gene Peptides

Antibodies directed against wild type or mutant fingerprint genepeptides, which are discussed, above, in Section 5.4.3, may also be usedas cardiovascular disease diagnostics and prognostics, as described, forexample, herein. Such diagnostic methods, may be used to detectabnormalities in the level of fingerprint gene protein expression, orabnormalities in the structure and/or tissue, cellular, or subcellularlocation of fingerprint gene protein. Structural differences mayinclude, for example, differences in the size, electronegativity, orantigenicity of the mutant fingerprint gene protein relative to thenormal fingerprint gene protein.

Protein from the tissue or cell type to be analyzed may easily bedetected or isolated using techniques which are well known to those ofskill in the art, including but not limited to western blot analysis.For a detailed explanation of methods for carrying out western blotanalysis, see Sambrook et al, 1989, supra, at Chapter 18. The proteindetection and isolation methods employed herein may also be such asthose described in Harlow and Lane, for example, (Harlow, E. and Lane,D., 1988, "Antibodies: A Laboratory Manual", Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), which is incorporatedherein by reference in its entirety.

Preferred diagnostic methods for the detection of wild type or mutantfingerprint gene peptide molecules may involve, for example,immunoassays wherein fingerprint gene peptides are detected by theirinteraction with an anti-fingerprint gene specific peptide antibody.

For example, antibodies, or fragments of antibodies, such as thosedescribed, above, in Section 5.4.3, useful in the present invention maybe used to quantitatively or qualitatively detect the presence of wildtype or mutant fingerprint gene peptides. This can be accomplished, forexample, by immunofluorescence techniques employing a fluorescentlylabeled antibody (see below) coupled with light microscopic, flowcytometric, or fluorimetric detection. Such techniques are especiallypreferred if the fingerprint gene peptides are expressed on the cellsurface.

The antibodies (or fragments thereof) useful in the present inventionmay, additionally, be employed histologically, as in immunofluorescenceor immunoelectron microscopy, for in situ detection of fingerprint genepeptides. In situ detection may be accomplished by removing ahistological specimen from a patient, and applying thereto a labeledantibody of the present invention. The antibody (or fragment) ispreferably applied by overlaying the labeled antibody (or fragment) ontoa biological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the fingerprint gene peptides, butalso their distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

Immunoassays for wild type or mutant fingerprint gene peptides typicallycomprise incubating a biological sample, such as a biological fluid, atissue extract, freshly harvested cells, or cells which have beenincubated in tissue culture, in the presence of a detectably labeledantibody capable of identifying fingerprint gene peptides, and detectingthe bound antibody by any of a number of techniques well known in theart.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled fingerprint genespecific antibody. The solid phase support may then be washed with thebuffer a second time to remove unbound antibody. The amount of boundlabel on solid support may then be detected by conventional means.

By "solid phase support or carrier" is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-wild type or mutantfingerprint gene peptide antibody may be determined according to wellknown methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

One of the ways in which the fingerprint gene peptide-specific antibodycan be detectably labeled is by linking the same to an enzyme and use inan enzyme immunoassay (EIA) (Voller, "The Enzyme Linked ImmunosorbentAssay (ELISA)", Diagnostic Horizons 2:1-7, 1978, MicrobiologicalAssociates Quarterly Publication, Walkersville, Md.; Voller, et al., J.Clin. Pathol. 31:507-520 (1978); Butler, Meth. Enzymol. 73:482-523(1981); Maggio, (ed.) Enzyme Immunoassay, CRC Press, Boca Raton, Fla.,1980; Ishikawa, et al., (eds.) Enzyme Immunoassay, Kgaku Shoin, Tokyo,1981). The enzyme which is bound to the antibody will react with anappropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-lycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase and cetylcholinesterase.The detection can be accomplished by colorimetric methods which employ achromogenic substrate for the enzyme. Detection may also be accomplishedby visual comparison of the extent of enzymatic reaction of a substratein comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild typeor mutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986, which is incorporated by reference herein). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵² Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

5.8.3. Imaging Cardiovascular Disease Conditions

In some cases, differentially expressed gene products identified hereinmay be up-regulated under cardiovascular disease conditions andexpressed on the surface of the affected tissue. Such target geneproducts allow for the non-invasive imaging of damaged or diseasedcardiovascular tissue for the purposed of diagnosis and directing oftreatment of the disease. For example, such differentially expressedgene products may include but are not limited to atherosclerosisspecific adhesion molecules responsible for atherogenesis, or monocytescavenger receptors that are up-regulated in response to oxidized LDL,which are discussed in Section 2, above. Alternatively, other suchsurface proteins may be specifically up-regulated in tissues sufferingfrom ischemia/reperfusion or other tissues with atherosclerotic orrestenotic lesions

As described in the example in Section 6, below, fchd602 is a gene thatis up-regulated in monocytes under disease conditions. Furthermore, thefchd602 gene encodes a novel protein containing multiple transmembranedomains. Not only is the fchd602 gene expressed in monocytes, which playa role in the initiation and progression of atherosclerotic lesions, itis also upregulated in monocytes under such disease conditions. Thefchd602 gene product, therefore, provides and excellent tool for imagingcardiovascular disease conditions.

This method can be applied in a similar manner to other transmembranetarget gene products, such as the fchd545 gene product. As described inthe example in Section 7, below, the fchd545 gene encodes a novel anionchannel, containing multiple transmembrane domains. Because the fchd545gene product might be more readily detected in normal tissue, as opposedto tissue in the disease state, it also provides an excellent tool forimaging cardiovascular disease conditions.

An example illustrating the use of this method in accordance with theinvention is provided in Section 9, below. Monoclonal and polyclonalantibodies, as described in Section 5.6.1.2, above, which specificallybind to such surface proteins, such as the fchd602 and fchd545 geneproducts, can be used for the diagnosis of cardiovascular disease by invivo tissue imaging techniques. Such antibodies raised against thefchd545 gene product are described in detail in the example in Section10, below. An antibody specific for a target gene product, or preferablyan antigen binding fragment thereof, is conjugated to a label (e.g., agamma emitting radioisotope) which generates a detectable signal andadministered to a subject (human or animal) suspected of havingcardiovascular disease. After sufficient time to allow thedetectably-labeled antibody to localize at the diseased or damagedtissue site (or sites), the signal generated by the label is detected bya photoscanning device. The detected signal is then converted to animage of the tissue. This image makes it possible to localize the tissuein vivo. This data can then be used to develop an appropriatetherapeutic strategy.

Antibody fragments, rather than whole antibody molecules, are generallypreferred for use in tissue imaging. Antibody fragments accumulate atthe tissue(s) more rapidly because they are distributed more readilythan are entire antibody molecules. Thus an image can be obtained inless time than is possible using whole antibody. These fragments arealso cleared more rapidly from tissues, resulting in a lower backgroundsignal. See, e.g., Haber et al., U.S. Pat. No. 4,036,945,; Goldenberg etal., U.S. Pat. No. 4,331,647. The divalent antigen binding fragment(Fab')₂ and the monovalent Fab are especially preferred. Such fragmentscan be prepared by digestion of the whole immunoglobulin molecule withthe enzymes pepsin or papain according to any of several well knownprotocols. The types of labels that are suitable for conjugation to amonoclonal antibody for diseased or damaged tissue localization include,but are not limited to radiolabels (i.e., radioisotopes), fluorescentlabels and biotin labels.

Among the radioisotopes that can be used to label antibodies or antibodyfragments, gamma-emitters, positron-emitters, X-ray-emitters andfluorescence-emitters are suitable for localization. Suitableradioisotopes for labeling antibodies include Iodine-131, Iodine-123,Iodine-125, Iodine-126, Iodine-133, Bromine-77, Indium-111, Indium-113m,Gallium-67, Gallium-68, Ruthenium-95, Ruthenium-97, Ruthenium-103,Ruthenium-105, Mercury-107, Mercury-203, Rhenium-99m, Rhenium-105,Rhenium-101, Tellurium-121m, Tellurium-122m, Tellurium-125m,Thulium-165, Thulium-167, Thulium-168, Technetium-99m and Fluorine-18.The halogens can be used more or less interchangeably as labels sincehalogen-labeled antibodies and/or normal immunoglobulins would havesubstantially the same kinetics and distribution and similar metabolism.

The gamma-emitters Indium-111 and Technetium-99m are preferred becausethese radiometals are detectable with a gamma camera and have favorablehalf lives for imaging in vivo. Antibody can be labelled with Indium-111or Technetium-99m via a conjugated metal chelator, such as DTPA(diethlenetriaminepentaacetic acid). See Krejcarek et al., 1977,Biochem. Biophys. Res. Comm. 77:581; Khaw et al., 1980, Science 209:295;Gansow et al., U.S. Pat. No. 4,472,509; Hnatowich, U.S. Pat. No.4,479,930, the teachings of which are incorporated herein by reference.

Fluorescent compounds that are suitable for conjugation to a monoclonalantibody include fluorescein sodium, fluorescein isothiocyanate, andTexas Red sulfonyl chloride. See, DeBelder & Wik, 1975, CarbohydrateResearch 44:254-257. Those skilled in the art will know, or will be ableto ascertain with no more than routine experimentation, otherfluorescent compounds that are suitable for labeling monoclonalantibodies.

6. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN RESPONSETO PARADIGM A: IN VITRO FOAM CELL PARADIGM

According to the invention, differential display may be used to detectgenes that are differentially expressed in monocytes that were treatedso as to simulate the conditions under which foam cells develop duringatherogenesis. By use of Paradigm A, the novel genes fchd602 and fchd605were identified Both fchd602 and fchd605 are up-regulated under thedisease condition of treatment with oxidized LDL.

The fchd602 gene product contains multiple transmembrane domains, andhas sequence similarity to the rat Cl-6 gene, which is induced inregenerating rat liver, is insulin inducible, and also contains multipletransmembrane domains (Diamond, R. H., et al., 1993, J. Biol. Chem. 268:15185-15192). The fchd605 gene product has sequence similarity to themouse gly96 gene (Charles, C. H. et al., 1993, Oncogene 8: 797-801), andto EST T49532.

The discovery of the up-regulation of these two genes provides afingerprint profile, e.g., markers, for monocytes in the process of foamcell formation. This profile can be used in the treatment and diagnosisof cardiovascular disease, including but not limited to atherosclerosis,ischemia/reperfusion, hypertension, restenosis, and arterialinflammation.

Furthermore, as a transmembrane protein, the fchd602 gene product can bereadily accessed or detected on the monocyte cell surface by othercompounds. It provides, therefore, an excellent target for detection ofcardiovascular disease states in diagnostic systems, as well as in themonitoring of the efficacy of compounds in clinical trials. Furthermore,the extracellular domains of this gene product provide targets whichallow for the design of especially efficient screening systems foridentifying compounds that bind to them. Such compounds can be useful intreating cardiovascular disease by modulating the activity of thetransmembrane gene product.

6.1. Materials and Methods

6.1.1. Cell Isolation and Culturing

Blood (˜200 ml) was drawn into chilled 20 ml vacutainer tubes to which 3ml of citrate phosphate dextrose (Sigma) was added. Blood was thenpooled into 50 ml tubes and spun in the Beckman GS-6R at 1250 RPM for 15minutes at 4° C. The upper clear layer (˜25 ml) was then removed with apipette and discarded and replaced with the same volume of 4° C. PBS.The blood was then mixed, and spun again at 2680 RPM for 15 minutes at4° C. The upper layer was then removed and discarded, and the buffy coatat the interface was removed in ˜5 ml and placed in a separate 50 mltube, and the pipette was washed with 20 ml PBS. Cells were added to a Tflask and stored at 4° C. for 16 hours. A small aliquot of the cellswere then removed and counted using a hemacytometer. The final red bloodcell concentration in the buffy coat population was then adjusted to1.5×10⁹ /ml with PBS, the cells were added to Leucoprep tubes (BectonDickinson) after being allowed to come to room temperature, and spun at2300 RPM for 25 minutes at 25° C. The upper clear layer was removed anddiscarded and the turbid layer over the gel was removed and pooled in 50ml tubes Samples were then diluted to 50 ml with PBS (25° C.) and spunat 1000 RPM for 10 minutes. The supernatant was then removed, and thepellet was resuspended in 50 ml PBS. This procedure was repeated 3 moretimes. After the last spin, the cells were resuspended in a small volumeof PBS and counted.

Tissue culture dishes were coated with bovine collagen before monocyteswere plated out. 1/6 volume of 7× RPHI (JRH Biosciences) was added toVitrogen 100 collagen (Celtrix) which was then diluted 1:10 with RPMI toa final concentration of 0.35 mg/ml. Collagen mixture was then added toplates (2.5 ml/100 mm dish) and placed at 37° C. for at least one hourto allow for gel formation. After gel formation has taken place, theRPMI was removed and cells were added in RPMI/10% plasma derived serum(PDS). PDS was prepared by drawing blood into chilled evacuated tubescontaining 1/10th volume 3.8% sodium citrate. Blood was then transferredinto new Sorvall tubes and spun at 14,000-16,000 RPM for 20 minutes at4° C. Plasma layer was removed and pooled in new tubes to which 1/50thvolume 1 M CaCl₂ was added. Plasma was mixed and aliquoted into newSorvall tubes and incubated at 37% for 2 hours to allow for fibrin clotformation. The clot was then disturbed with a pipette to allow it tocontract and tubes were spun at 14,500 RPM for 20 minutes at 25° C.Supernatant was collected, pooled, and heat inactivated at 56° C. priorto sterile filtration and freezing.

Purified human monocytes were cultured in 10% PDS/RPMI containing 5units/ml of Genzyme recombinant human MCSF for 5 days before beingtreated with LDL, oxidized LDL, acetylated LDL (all LDL at 50 μg/ml),lysophosphatidylcholine (Sigma, 37.5 μM), or homocysteine (Sigma, 1 mM)After incubation with these reagents for periods ranging from 2 hours upto 3 days, the media was withdrawn and the cells were dissolved in RNAlysis buffer and RNA was prepared as described, above, in Section 6.1.

Lipoproteins For oxidation, human LDL (Sigma) was first diluted to 1mg/ml with PBS and then dialyzed against PBS at 4° C. overnight. LDL wasthen diluted to 0.3 mg/ml with PBS. CuSO₄.5H₂ O was then added to 5 uMfinal concentration, and the solution was incubated in a T flask in a37° C. incubator for 24 hr. LDL solution was then dialyzed at 4° C.against 0.15 M NaCl/0.3 mM EDTA for 2 days with several changes, beforebeing removed and concentrated using an Amicon spin column by spinningfor 1 hr. 4000 RPM at 4° C.

For acetylation, 1 ml of 5 mg/ml LDL was added to 1 ml of a saturatedsolution of NaOAc in a 15 ml tube on ice on a shaker at 4° C. 8 μl ofacetic anhydride was added 2 μl at a time over 1 hr. LDL was thendialyzed for 48 hr. against 0.15 M NaCl/0.3 mM EDTA at 4° C. for 48 hr.with several changes. Final concentrations of derivatized LDL's weredetermined by comparing to a dilution curve of native LDL analyzed atOD₂₈₀, with 0.15 M NaCl/0.3 mM EDTA used as diluent in all cases.

6.1.2. Analysis of Paradigm Material

Differential Display

Removal of DNA: The RNA pellet was resuspended in H₂ O and quantified byspectrophotometry at OD₂₆₀. Approximately half of the sample was thentreated with DNAse I to remove contaminating chromosomal DNA. RNA wasamplified by PCR using the following procedure. 50 ul RNA sample (10-20μg), 5.7 μl 10×PCR buffer (Perkin-Elmer/Cetus), 1 μl RNAse inhibitor (40units/μl) (Boehringer Mannheim, Germany) were mixed together, vortexed,and briefly spun. 2 μl DNAse I (10 units/μl) (Boehringer Mannheim) wasadded to the reaction which was incubated for 30 min. at 37° C. Thetotal volume was brought to 200 μl with DEPC H₂ O, extracted once withphenol/chloroform, once with chloroform, and precipitated by adding 20μl 3 M NaOAc, pH 4.8, (DEPC-treated), 500 μl absolute ETOH andincubating for 1 hour on dry ice or -20° C. overnight. The precipitatedsample was centrifuged for 15 min., and the pellet was washed with 70%ETOH. The sample was re-centrifuged, the remaining liquid was aspirated,and the pellet was resuspended in 100 μl H₂ O. The concentration of RNAwas measured by reading the OD₂₆₀.

First strand cDNA synthesis: For each RNA sample duplicate reactionswere carried out in parallel. 400 ng RNA plus DEPC H₂ O in a totalvolume of 10 μl were added to 4 μl T₁₁ XX reverse primer (10 μM)(Operon). The specific primers used in each experiment are provided inthe Description of the Figures in Section 4, above. The mixture wasincubated at 70° C. for 5 min. to denature the RNA and then placed atr.t. 26 μl of reaction mix containing the following components was addedto each denatured RNA/primer sample: 8 μl 5× First Strand Buffer(Gibco/BRL, Gaithersburg, Md.), 4 μl 0.1 M DTT (Gibco/BRL), 2 μl RNAseinhibitor (40 units/μl) (Boehringer Mannheim) , 4 μl 200 μM dNTP mix, 6μl H₂ O, 2 μl Superscript reverse transcriptase (200 units/μl)(Gibco/BRL). The reactions were mixed gently and incubated for 30 min.at 42° C. 60 μl of H₂ O (final volume=100 μl) were then added and thesamples were denatured for 5 min. at 85° C. and stored at -20° C.

PCR reactions: 13 μl of reaction mix was added to each tube of a 96 wellplate on ice. The reaction mix contained 6.4 μl H₂ O, 2 μl 10× PCRBuffer (Perkin-Elmer), 2 μl 20 μM dNTP's, 0.4 μl ³⁵ S dATP (12.5 μCi/μl;50 μCi total) (Dupont/NEN), 2 μl forward (for-) primer (10 μM) (Operon),and 0.2 μl AmpliTaq Polymerase (5 units/μl) (Perkin-Elmer).

Next, 2 μl of reverse (rev-) primer (T₁₁ XX, 10 μM) were added to theside of each tube followed by 5 μl of cDNA also to the sides of thetubes, which were still on ice. The specific primers used in eachexperiment were as follows:

fchd602: rev-T₁₁ XC (SEQ ID NO:13) and for-GTGAGGCGTC (SEQ ID NO:14)

fchd605: rev-T₁₁ XC (SEQ ID NO:13) and for-TGGACCGGTG (SEQ ID NO:15)

Tubes were capped and mixed, and brought up to 1000 RPM in a centrifugethen returned immediately to ice. The PCR machine (Perkin-Elmer 9600)was programmed for differential display as follows:

    ______________________________________                                         94° C.                                                                              2                min.                                             *94° C. 15 sec.                                                        *40° C. 2 min.                                                         *ramp 72° C. 1 min.                                                    *72° C. 30 sec.                                                         72° C. 5 min.                                                          4° C.  hold                                                         ______________________________________                                         * = X40                                                                  

When the PCR machine reached 94° C., the plate was removed from ice andplaced directly into the Perkin-Elmer 9600 PCR machine. Following PCR,15 μl of loading dye, containing 80% formamide, 10 mM EDTA, 1 mg/mlxylene cyanol, 1 mg/ml bromphenol blue were added. The loading dye andreaction were mixed, incubated at 85° C. for 5 min., cooled on ice,centrifuged, and placed on ice. Approximately 4 μl from each tube wereloaded onto a prerun (60V) 6% acrylamide gels The gel was run atapproximately 80V until top dye front was about 1 inch from bottom. Thegel was transferred to 3 MM paper (Whatman Paper, England) and driedunder vacuum. Bands were visualized by autoradiography.

Band isolation and amplification: Differentially expressed bands wereexcised from the dried gel with a razor blade and placed into amicrofuge tube with 100 μl H₂ O and heated at 100° C. for 5 min.,vortexed, heated again to 100° C. for 5 min., and vortex again. Aftercooling, 100 μl H₂ O, 20 μl 3 M NaOAc, 1 μl glycogen (20 mg/ml), and 500μl ethanol were added and chilled. After centrifugation, the pellet waswashed and resuspended in 10 μl H₂ O.

The isolated differentially expressed bands were then amplified by PCRusing the following reaction conditions:

    ______________________________________                                        58           μl    H.sub.2 O                                                 10 μl 10x PCR Buffer                                                       10 μl 200 μm dNTP's                                                     10 μl 10 μM reverse primer                                              10 μl 10 μM forward primer                                              1.5 μl amplified band                                                      0.5 μl AmpliTaq polymerase (5 units/μl)                                   (Perkin Elmer)                                                            ______________________________________                                    

PCR was performed using the program described in this Section, above,for differential display. After PCR, glycerol loading dyes were addedand samples were loaded onto a 2% preparative TAE/Biogel (Bio101, LaJolla, Calif.) agarose gel and eluted. Bands were then excised from thegel with a razor blade and vortexed for 15 min. at r.t., and purifiedusing the Mermaid kit from Bio101 by adding 3 volumes of Mermaid highsalt binding solution and 8 μl of resuspended glassfog in a microfugetube. Glassfog was then pelleted, washed 3 times with ethanol washsolution, and then DNA was eluted twice in 10 μl at 50° C.

Subcloning: The TA cloning kit (Invitrogen, San Diego, Calif.) was usedto subclone the amplified bands. The ligation reaction typicallyconsisted of 4 μl sterile H₂ O, 1 μl ligation buffer, 2 μl TA cloningvector, 2 μl PCR product, and 1 μl T4 DNA ligase. The volume of PCRproduct can vary, but the total volume of PCR product plus H₂ O wasalways 6 μl. Ligations (including vector alone) were incubated overnightat 12° C. before bacterial transformation. TA cloning kit competentbacteria (INVαF': enda1, recAl, hsdR17(r-k, m+k), supE44, λ-, thi-1,gyrA, relA1, φ80lacZαΔM15Δ(lacZYA-argF), deoR+, F') were thawed on iceand 2 μl of 0.5 M β-mercaptoethanol were added to each tube. 2 μl fromeach ligation were added to each tube of competent cells (50 μl), mixedwithout vortexing, and incubated on ice for 30 min.

Tubes were then placed in 42° C. bath for exactly 30 sec., before beingreturned to ice for 2 min. 450 μl of SOC media (Sambrook et al., 1989,supra) were then added to each tube which were then shaken at 37° C. for1 hr. Bacteria were then pelleted, resuspended in ˜200 μl SOC and platedon Luria broth agar plates containing X-gal and 60 μg/ml ampicillin andincubated overnight at 37° C. White colonies were then picked andscreened for inserts using PCR.

A master mix containing 2 μl 10× PCR buffer, 1.6 μl 2.5 mM dNTP's, 0.1 l25 mM MgCl₂, 0.2 μl M13 reverse primer (100 ng/μl), 0.2 μl M13 forwardprimer (100 ng/μl), 0.1 μl AmpliTaq (Perkin-Elmer), and 15.8 μl H₂ O wasmade. 40 μl of the master mix were aliquoted into tubes of a 96 wellplate, and whole bacteria were added with a pipette tip prior to PCR.The PCR machine (Perkin-Elmer 9600) was programmed for insert screeningas follows:

    ______________________________________                                         94° C.                                                                               2               min.                                             *94° C. 15 sec.                                                        *47° C. 2 min.                                                         *ramp 72° C. 30 sec.                                                   *72° C. 30 sec.                                                         72° C. 10 min.                                                         4° C.  hold                                                         ______________________________________                                         * = X35                                                                  

Reaction products were eluted on a 2% agarose gel and compared to vectorcontrol. Colonies with vectors containing inserts were purified bystreaking onto LB/Amp plates. Vectors were isolated from such strainsand subjected to sequence analysis, using an Applied BiosystemsAutomated Sequencer (Applied Biosystems, Inc. Seattle, Wash.).

Northern analysis: Northern analysis was performed to confirm thedifferential expression of the genes corresponding to the amplifiedbands. The probes used to detect mRNA were synthesized as follows:typically 2 μl amplified band (˜30 ng), 7 μl H₂ O, and 2 μl 10×Hexanucleotide mix (Boehringer-Mannheim) were mixed and heated to 95° C.for 5 min., and then allowed to cool on ice. The volume of the amplifiedband can vary, but the total volume of the band plus H₂ O was always 9μl. 3 μl dATP/dGTP/dTTP mix (1:1:1 of 0.5 mM each), 5 μl α³² p dCTP 3000Ci/mM (50 μCi total) (Amersham, Arlington Heights, Ill.), and 1 μlKlenow (2 units) (Boehringer-Mannheim) were mixed and incubated at 37°C. After 1 hr, 30 μl TE were added and the reaction was loaded onto aBiospin-6™ column (Biorad, Hercules, Calif.), and centrifuged. A 1 μlaliquot of eluate was used to measure incorporation in a scintillationcounter with scintillant to ensure that 10⁶ cpm/μl of incorporation wasachieved

The samples were loaded onto a denaturing agarose gel. A 300 ml 1% gelwas made by adding 3 g of agarose (SeaKem™ LE, FMC BioProducts,Rockland, Me.) and 60 ml of 5× MOPS buffer to 210 ml sterile H2O. 5×MOPS buffer (0.1 M MOPS (pH 7.0), 40 mM NaOAc, 5 mM EDTA (pH 8.0)) wasmade by adding 20.6 g of HOPS to 800 ml of 50 mM NaOAc (13.3 ml of 3 MNaOAc pH 4.8 in 800 ml sterile H₂ O); then adjusting the pH to 7.0 with10 M NaOH; adding 10 ml of 0.5 M EDTA (pH 8.0); and adding H₂ O to afinal volume of 1 L. The mixture was heated until melted, then cooled to50° C., at which time 5 μl ethidium bromide (5 mg/ml) and 30 ml of 37%formaldehyde of gel were added. The gel was swirled quickly to mix, andthen poured immediately.

2 μg RNA sample, 1× final 1.5× RNA loading dyes (60% formamide, 9%formaldehyde, 1.5× MOPS, 0.075% XC/BPB dyes) and H₂ O were mixed to afinal volume of 40 μl. The tubes were heated at 65° C. for 5 min. andthen cooled on ice. 10 μg of RNA MW standards (New England Biolabs,Beverly, Mass.) were also denatured with dye and loaded onto the gel.The gel was run overnight at 32V in MOPS running buffer.

The gel was then soaked in 0.5 μg/ml Ethidium Bromide for 45 min., 50 mMNaOH/0.1 M NaCl for 30 min., 0.1 M Tris pH 8.0 for 30 min., and 20× SSCfor 20 min. Each soaking step was done at r.t. with shaking. The gel wasthen photographed along with a fluorescent ruler before blotting withHybond-N membrane (Amersham), according to the methods of Sambrook etal., 1989, supra, in 20× SSC overnight.

Northern blot hybridizations were carried out as follows: forpre-hybridization, the blot was placed into roller bottle containing 10ml of rapid-hyb solution (Amersham), and placed into 65° C. incubatorfor at least 1 hr. For hybridization, 1×10⁷ cpm of the probe was thenheated to 95° C., chilled on ice, and added to 10 ml of rapid-hybsolution. The prehybridization solution was then replaced with probesolution and incubated for 3 hr at 65° C. The following day, the blotwas washed once for 20 min. at r.t. in 2× SSC/0.1% SDS and twice for 15min. at 65° C. in 0.1× SSC/0.1% SDS before being covered in plastic wrapand put down for exposure.

RT-PCR Analysis: RT-PCR was performed to detect differentially expressedlevels of mRNA from the genes corresponding to amplified bands. Firststrand synthesis was conducted by mixing 20 μl DNased RNA (˜2 μg), 1 μloligo dT (Operon) (1 μg), and 9.75 μl H₂ O. The samples were heated at70° C. for 10 min., and then allowed to cool on ice. 10 μl first strandbuffer (Gibco/BRL), 5 μl 0.1 M DTT, 1.25 μl 20 mM dNTP's (500 μM final),1 μl RNAsin (40 units/μl) (Boehringer Mannheim) and 2 μl SuperscriptReverse Transcriptase (200 units/μl) (Gibco/BRL) were added to thereaction, incubated at 42° C. for 1 hr., and then placed at 85° C. for 5min., and stored at -20° C.

PCR was performed on the reverse transcribed samples. Each reactioncontained 2 μl 10× PCR buffer, 14.5 μl H₂ O, 0.2 μl 20 mM dNTP's (200 μMfinal), 0.5 μl 20 μM forward primer (0.4 μM final), 0.5 μl 20 μM reverseprimer (0.4 μM final), 0.3 μl AmpliTaq polymerase (Perkin-Elmer/Cetus),2 μl cDNA dilution or positive control (˜40 μg). The specific primersused in each experiment are provided in the Description of the Figuresin Section 4, above. Samples were placed in the PCR 9600 machine at 94°C. (hot start), which was programmed as follows:

    ______________________________________                                         94° C.  2         min. (samples loaded)                                 *94° C. 45 sec.                                                        *55° C. 45 sec.                                                        *72° C. 2 min.                                                          72° C. 5 min.                                                          4° C.  hold                                                         ______________________________________                                         * = 35x                                                                  

Reactions were carried out on cDNA dilution series and tubes wereremoved at various cycles from the machine during 72° C. step. Reactionproducts were eluted on a 1.8% agarose gel and visualized with ethidiumbromide.

Gene Retrieval: Amplified sequences, which contained portions of thegenes, were subcloned and then used individually to retrieve a cDNAencoding the corresponding gene. Probes were prepared by isolating thesubcloned insert DNA from vector DNA, and labeling with ³² p asdescribed above in Section 6.1.2. Labeled insert DNA containing fchd602sequences was used to probe a cDNA library prepared from humanmacrophage cell line U937. Labeled insert DNA containing fchd605sequences was used to probe a cDNA library prepared from human primaryblood monocytes. The cDNA libraries were prepared and screened accordingto methods routinely practiced in the art (see Sambrook et al., 1989,supra). Plaques from the libraries that were detected by the probes wereisolated and the cDNA insert within the phage vector was sequenced.

The RACE procedure kit was used either as an alternative to cDNA libraryscreening, or, when the cDNA library did not yield a clone encoding thefull-length gene, to obtain adjacent sequences of the gene. Theprocedure was carried out according to the manufacturer's instructions(Clontech, Palo Alto, Calif.; see also: Chenchik, et al., 1995,CLONTECHniques (X) 1: 5-8; Barnes, 1994, Proc. Natl. Acad. Sci. USA 91:2216-2220; and Cheng et al., Proc. Natl. Acad. Sci. USA 91: 5695-5699).Primers were designed based either on amplified sequences, or onsequences obtained from isolates from the cDNA libraries. Template mRNAfor fchd605 was isolated from human primary blood monocytes.

6.1.3. Chromosomal Localization of Target Genes

Once the nucleotide sequence has been determined, the presence of thegene on a particular chromosome is detected Oligonucleotide primersbased on the nucleotide sequence of the target gene are used in PCRreactions using individual human chromosomes as templates. Individualsamples of each the twenty-three human chromosomes are commerciallyavailable (Coriel Institute for Medical Research, Camden, N.J.). Thechromosomal DNA is amplified according to the following conditions: 10ng chromosomal DNA, 2 μl 10×PCR buffer, 1.6 μl 2.5 mM dNTP's, 0.1 μl 25mM NgCl₂, 0.2 μl reverse primer (100 ng/μl), 0.2 μl forward primer (100ng/μl), 0.1 μl Taq polymerase, and 15.8 μl H₂ O. Samples are placed inthe PCR 9600 machine at 94° C. (hot start), which is programmed asfollows:

    ______________________________________                                         94° C. 2          min. (samples loaded)                                 *94° C. 20 sec.                                                        *55° C. 30  sec.                                                       *72° C. 30  sec.                                                        72° C. 5  min.                                                         4° C.   hold                                                        ______________________________________                                         * = 35x                                                                  

6.2. Results

Differential display was performed on monocytes treated with oxidizedLDL and untreated monocytes. Bands corresponding to fchd602 and fchd605were detected as up-regulated by oxidized LDL, as compared with theuntreated monocytes. The up-regulation was confirmed by northern blotanalysis.

The fchd602 gene produced a 2.5 kb mRNA that was up-regulated after 5hours of treatment with oxidized LDL, minimally oxidized LDL, andlysophosphatidylcholine. No message was detected in untreated or nativeLDL treated control monocytes. The amplified DNA sequence was used torecover a cDNA of approximately 875 bp comprising an open reading frameencoding approximately 182 amino acids. The DNA sequence and encodedamino acid sequence of this cDNA from the fchd602 gene is shown in FIG.4. The open reading frame has 88% sequence similarity to the rat Cl-6gene, which is induced in regenerating rat liver, is insulin inducible,and also contains multiple transmembrane domains (Diamond, R. H., etal., 1993, J. Biol. Chem. 268: 15185-15192).

The fchd605 gene produced a 1.5 kb mRNA that is up-regulated after 5hours treatment with oxidized LDL, and to a lesser degree with nativeLDL, as compared to untreated monocytes. The amplified DNA was sequencedand used to recover a cDNA of approximately 2.2 kb, which was sequencedto reveal a partial open reading frame of approximately 258 bp, encodingapproximately 86 amino acids. The DNA sequence and encoded amino acidsequence from the fchd605 gene is shown in FIG. 5. The sequence hassimilarity to the mouse gly96 gene, which encodes a cytokine inducibleglycosylated protein expressed in mouse lung, testes, and uterus.

7. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN RESPONSETO PARADIGM D: ENDOTHELIAL CELL SHEAR STRESS

According to the invention, differential display was used to detectgenes that are differentially expressed in endothelial cells that weresubjected to fluid shear stress in vitro. Shear stress is thought to beresponsible for the prevalence of atherosclerotic lesions in areas ofunusual circulatory flow. Using the method of Paradigm D, three novelDNA sequences were identified.

The fchd531 gene is down-regulated in endothelial cells under bothturbulent and laminar shear stress, as compared to the static control.The fchd531 gene encodes a novel 570 amino acid polypeptide, and has 94%sequence similarity to the mouse penta zinc finger gene (Pzf), which hasnot been published, but is contained in the GenBank sequence data baseunder accession no U05343.

The fchd540 gene is up-regulated in endothelial cells under laminarshear stress, but is not up-regulated by IL-1 treatment. The fchd540gene encodes a novel intracellular protein which has sequence similarityto the Drosophila Mad protein (Sekelsky et al., 1995, Genetics 139:1347-1358).

The fchd545 gene is down-regulated in endothelial cells under laminarshear stress as compared to endothelial cells under turbulent shearstress and static control endothelial cells. The fchd545 gene encodes an848 amino acid polypeptide which has 73% sequence similarity to thehuman Voltage-dependent Anion Channel protein (Blachly-Dyson, E., etal., 1993, J. Biol. Chem. 268: 1835-1841.). The fchd545 gene is alsoexpressed in the human heart, smooth muscles, and testes.

The up-regulation of the fchd540 gene and down-regulation of the fchd531and fchd545 genes in shear stressed endothelial cells provides afingerprint for the study of cardiovascular diseases, including but notlimited to atherosclerosis, ischemia/reperfusion, hypertension andrestenosis. The fact that one of these genes, fchd540, is notup-regulated under Paradigm C (IL-1 induction) provides an extremelyuseful means of distinguishing and targeting physiological phenomenaspecific to shear stress.

Furthermore, as a transmembrane protein, the fchd545 gene product can bereadily accessed or detected on the endothelial cell surface by othercompounds. It provides, therefore, an excellent target for detection ofcardiovascular disease states in diagnostic systems, as well as in themonitoring of the efficacy of compounds in clinical trials. Furthermore,the extracellular domains of this gene product provide targets whichallow for designing especially efficient screening systems foridentifying compounds that bind to them. Such compounds can be useful intreating cardiovascular disease by modulating the activity of thetransmembrane gene product.

7.1. Materials and Methods

Primary cultures of HUVEC's were established from normal term umbilicalcords as described (In Progress in Hemostasis and Thrombosis, Vol. 3, P.Spaet, editor, Grune & Stratton Inc., New York, 1-28). Cells were grownin 20% fetal calf serum complete media (1989, J. Immunol. 142:2257-2263) and passaged 1-3 times before shear stress induction.

For induction, second passage HUVEC's were plated on tissueculture-treated polystyrene and subjected to 10 dyn/cm² laminar flow for1 and 6 hr. as described (1994, J. Clin. Invest. 94: 885-891) or 3-10dyn/cm² turbulent flow as previously described (1986 Proc. Natl Acad.Sci. U.S.A. 83: 2114-2117). RNA was isolated as described, above, inSection 6.1. Differential display, Northern analysis, RT-PCR,subcloning, and DNA sequencing were carried out as described, above, inSection 6.1.2. Specific primers used in differential display were asfollows:

fchd531: for-T₁₁ XA (SEQ ID NO:16) and rev-AGACGTCCAC (SEQ ID NO:17)

fchd540: for-T₁₁ XA (SEQ ID NO:16) and rev-ACTTCGCCAC (SEQ ID NO:18)

fchd545: for-T₁₁ XC (SEQ ID NO:13) and rev-TCGGACGTGA (SEQ ID NO:19)

Amplified sequences, which contained portions of the genes, weresubcloned and then used individually to retrieve a cDNA encoding thecorresponding gene. Probes were prepared by isolating the subclonedinsert DNA from vector DNA, and labeling with ³² p as described above inSection 6.1.2. Labeled insert DNA was used to probe cDNA libraryprepared from shear stress induced endothelial cells. The library wasprepared and probed using methods routinely practiced in the art (seeSambrook et al., 1989, supra). Plaques from the libraries that weredetected by the probes were isolated and the cDNA insert within thephage vector was sequenced.

The RACE procedure kit was used either as an alternative to cDNA libraryscreening, or, when the cDNA library did not yield a clone encoding thefull-length gene, to obtain adjacent sequences of the gene. Theprocedure was carried out according to the manufacturer's instructions(Clontech, Palo Alto, Calif.; see also: Chenchik, et al., 1995,CLONTECHniques (X) 1: 5-8; Barnes, 1994, Proc. Natl. Acad. Sci. USA 91:2216-2220; and Cheng et al., Proc. Natl. Acad. Sci. USA 91: 5695-5699).Primers were designed based either on amplified sequences, or onsequences obtained from isolates from the cDNA libraries. Template mRNAwas isolated from shear stressed HUVEC's.

Northern blot analysis of RNA extracted from various human organs andtissues was performed using commercially available pre-blotted filters(Clontech, Palo Alto, Calif.).

7.2. Results

An amplified fchd531 fragment obtained from differential display wassubcloned and sequenced, and used to obtain a 1.9 kb cDNA containing theentire fchd531 coding region. The DNA sequence and encoded amino acidsequence of the novel fchd531 gene is shown in FIGS. 1A-C. The fchd531gene encodes a 570 amino acid polypeptide, and has 94% sequencesimilarity to the mouse penta zinc finger gene (Pzf) (GenBank accessionnumber U05343). Northern analysis of HUVEC's which were subjectedturbulent and laminar shear stress demonstrated that the fchd531 geneproduces an approximately 5 kb message which is down-regulated underlaminar shear stress, but not turbulent shear stress, compared with thestatic control.

The fchd540 gene was detected as an up-regulated message under shearstress The amplified fragment was used to probe a Northern blotcontaining samples from HUVECs treated with laminar shear stress. A 4.4kb fchd540 mRNA is up-regulated after 6 hours treatment with laminarshear stress. The fchd540 gene is not induced by IL-1 by the method ofParadigm C, (Section 5.1.1.5, above). The amplified fragment wassequenced and used to obtain a 2.7 kb cDNA containing the entire fchd540coding region. The DNA sequence and encoded amino acid sequence from thefchd540 gene is shown in FIGS. 2A-2C. The fchd540 gene encodes a 426amino acid polypeptide and has sequence similarity to the Drosophila Madgene (Sekelsky et al., 1995, Genetics 139: 1347-1358).

The fchd545 gene was detected as a down-regulated message under shearstress. Northern analysis revealed that the fchd545 gene produces a 1.4kb message which is down regulated by turbulent shear stress, but not bylaminar shear stress, as compared with static control. The amplifiedfragment was sequenced and used to isolate a 1.4 kb cDNA containing thecomplete fchd545 coding sequence. The DNA sequence and encoded aminoacid sequence of the fchd545 gene is shown in FIGS. 3A-3B. The fchd545gene encodes a 283 amino acid polypeptide which has 73% sequencesimilarity to the human Voltage-dependent Anion Channel (Blachly-Dyson,E., et al., 1993, J. Biol. Chem. 268: 1835-1841). Northern analysis of acommercially available (Clontech, Palo Alto, Calif.) northern blotrevealed that the fchd545 gene is expressed in human heart, smoothmuscle, and testes.

8. EXAMPLE: USE OF GENES UNDER PARADIGM A AS SURROGATE MARKERS INCLINICAL TRIALS

According to the invention, the fingerprint profile derived from any ofthe paradigms described in Sections 5.1.1.1 through 5.1.1.6 may be usedto monitor clinical trials of drugs in human patients. The fingerprintprofile, described generally in Section 5.5.4, above, indicates thecharacteristic pattern of differential gene regulation corresponding toa particular disease state. Paradigm A, described in Section 5.1.1.1,and illustrated in the example in Section 6, above, for example,provides the fingerprint profile of monocytes under oxidative stress.The target genes, therefore, serve as surrogate markers by giving anindicative reading of the physiological response of monocytes to theuptake of oxidized LDL. Accordingly, the influence of anti-oxidant drugson the oxidative potential may be measured by performing differentialdisplay on the monocytes of patients undergoing clinical tests.

8.1. Treatment of Patients and Cell Isolation

Test patients may be administered compounds suspected of havinganti-oxidant activity. Control patients may be given a placebo.

Blood may be drawn from each patient after a 12 hour period of fastingand monocytes may be purified as described, above, in Section 7.1.1. RNAmay be isolated as described in Section 6.1.1, above. Primers may thenbe designed for amplification based on the DNA sequence of target genesidentified as up-regulated, such as fchd602 and fchd605, ordown-regulated under Paradigm A.

8.2. Analysis of Samples

RNA may be subjected to differential display analysis as described inSection 6.1.2, above. A decrease in the physiological response state ofthe monocytes is indicated by a decreased intensity of those bandscorresponding to fchd602 and fchd605, which were up-regulated byoxidized LDL under Paradigm A, as described in Section 6.2, above

9. EXAMPLE: IMAGING OF A CARDIOVASCULAR DISEASE CONDITION

According to the invention, differentially expressed gene products whichare localized on the surface of affected tissue may be used as markersfor imaging the diseased or damaged tissue. Conjugated antibodies thatare specific to the differentially expressed gene product may beadministered to a patient or a test animal intravenously. This methodprovides the advantage of allowing the diseased or damaged tissue to bevisualized non-invasively.

For the purposes of illustration, this method is described in detail forthe fchd602 gene product. The principles and techniques can be appliedto any transmembrane target gene product, including, for example, thefchd545 gene product.

9.1. Monoclonal Conjugated Antibodies

The differentially expressed surface gene product, such as the fchd602gene product, is expressed in a recombinant host and purified usingmethods described in Section 5.4.2, above. Preferably, a proteinfragment comprising one or more of the extracellular domains of thefchd602 product is produced. Once purified, it is be used to produceF(ab')₂ or Fab fragments, as described in Section 5.4.3, above. Thesefragments are then labelled with technetium-99m (^(99m) Tc) using aconjugated metal chelator, such as DTPA as described in section 5.8.3,above

9.2. Administration and Detection of Imaging Agents

Labeled MAb may be administered intravenously to a patient beingdiagnosed for atherosclerosis, restenosis, or ischemia/reperfusion.Sufficient time is allowed for the detectably-labeled antibody tolocalize at the diseased or damaged tissue site (or sites), and bind tothe fchd602 gene product. The signal generated by the label is detectedby a photoscanning device. The detected signal is then converted to animage of the tissue, revealing cells, such as monocytes, in whichfchd602 gene expression is up-regulated.

10. POLYCLONAL ANTIBODIES TO TARGET GENE PEPTIDE SEQUENCES

Peptide sequences corresponding to the indicated amino sequences ofcDNAs were selected and submitted to Research Genetics (Huntsville,Ala.) for synthesis and antibody production Peptides were modified asdescribed (Tam, J. P., 1988, Proc. Natl. Acad. Sci. USA 85: 5409-5413;Tam, J. P., and Zavala, F, 1989, J. Immunol. Methods 124: 53-61; Tam, J.P., and Lu, Y. A., 1989, Proc. Natl. Acad. Sci. USA 86: 9084-9088),emulsified in an equal volume of Freund's adjuvant and injected intorabbits at 3 to 4 subcutaneous dorsal sites for a total volume of 1.0 ml(0.5 mg peptide) per immunization. The animals were boosted after 2 and6 weeks and bled at weeks 4, 8, and 10. The blood was allowed to clotand serum was collected by centrifugation.

The peptides used are summarized below:

    ______________________________________                                        fchd545 Peptide Antigens                                                        Name      Position Sequence                                                 ______________________________________                                        fchd545.1                                                                             48-63    YTDTGKASGNLETKYK (SEQ ID NO: 43)                               fchd545.2 107-121 TGKKSGKLKASYKRD (SEQ ID NO: 44)                           ______________________________________                                    

11. EXAMPLE: THE RCHD534 AND FCHD540 GENE PRODUCTS INTERACT

The novel fchd540 gene and its nucleotide sequence is described inSection 7, above. The fchd540 gene shares homology with the DrosophilaMad gene. The rchd534 gene (described in Applicant's co-pendingapplication Ser. No. 08/485,573, filed Jun. 7, 1995, which isincorporated by reference in its entirety herein) is another gene thatis up-regulated in endothelial cells by shear stress. The DNA andencoded amino acid sequence of the rchd534 gene is shown in FIGS. 6A-6C.The rchd534 gene was deposited in the Agricultural Research ServiceCulture Collection (NRRL) in microorganism FCHD534 on Jun. 6, 1995 andassigned the NRRL Accession No. B-21459. The rchd534 gene also shareshomology with the Drosophila Mad gene. Mad genes have been shown to playa role in the TGF-β signalling pathway (Sekelsky et al., 1995, Genetics139: 1347-1358; Chen et al., 1996, Nature 383: 691-696; Serra, et al.,1996, Nature Medicine 2: 390-391) TGF-β signalling is considered to bebeneficial to atherosclerosis and restenosis (Border et al, 1995, NatureMedicine 1: 1000; Grainger, et al., 1995, Nature Medicine 1 1067-1073;Kojima, et al., 1991, J. Cell Biol. 113: 1439-1445; Nikol, et al., 1992,J. Clin. Invest. 90: 1582-1592).

The data described below demonstrate that the rchd534 and fchd540proteins interact with one another; and this interaction may lead to theinhibition of TGF-β signalling. Furthermore, the expression of these twogenes, as described below, is specific to endothelial cells. Becausethese two genes 1) are both expressed specifically in endothelial cells,2) are both up-regulated in endothelial cells under certain conditions,3) encode MAD proteins that interact with one another in endothelialcells, and 4) inhibit TGF-β signalling (which is considered to bebeneficial to atherosclerosis), rchd534 and fchd540 proteins areattractive targets for therapeutic intervention in cardiovasculardisease. In particular, treatment regimens that inhibit the interactionor activity of the rchd534 and fchd540 proteins can be beneficial forthe treatment cardiovascular disease.

Further analyses demonstrated that the rchd534 protein interacts withitself to form a homodimer. Thus, treatment regimens that inhibit theinteraction of the rchd534 protein with itself can be beneficial for thetreatment cardiovascular disease.

In addition, the analyses described below demonstrated novelinteractions of both the rchd534 and fchd540 proteins with otherproteins known to be involved in the TGF-β signalling pathway. Theprotein members of the TGF-β signalling pathway tested included MADR1(Hoodless et al., 1996, Cell 85:489-500), MADR2 (Eppert et al., 1996,Cell 86: 543-552), DPC4 (Raftery et al., 1988, Genetics 139: 241-254),TβRI, TSR1, ActRIb, ALK3, and ALK6 (Wieser et al., 1995, EMBO J. 14:2199-2208). For example, the rchd534 protein interacts strongly inendothelial cells with MADR1, MADR2, DPC4, and weakly in 293 (humanembryonic kidney) cells with activated forms of receptors TβRI andActRI. The fchd540 protein interacts strongly in 293 cells withactivated forms of receptors TβRI and ALK6.

In the absence of transfected rchd534 and fchd540 genes, transfectedMADR1 or transfected MADR2 mediated a 20-fold induction of aTGF-βinducible promoter in BAECs. Co-expression of either transfectedrchd534 or transfected fchd540 in this system eliminated the induction,and also prevented the localization of MADR2 in the nucleus in responseto TGF-β signalling. Therefore, treatment regimens that inhibit theinteraction of the rchd534 and fchd540 proteins with other proteinsinvolved in the TGF-β pathway also can be beneficial for the treatmentcardiovascular of disease. As described above, the expression of rchd534and fchd540 is specific, within arterial tissue, to endothelial cells.Accordingly, the rchd534 and fchd540 genes may be targets forintervention in a variety of inflammatory and fibroproliferativedisorders that involve endothelial cells, including, but not limited to,cancer angiogenesis, inflammation, and fibrosis.

11.1. Materials and Methods

11.1.1 Yeast Strains, Media, and Microbiological Techniques

Standard yeast media including synthetic complete medium lackingL-leucine, L-tryptophan, and L-histidine were prepared and yeast geneticmanipulations were performed as described (Sherman, 1991, Meth.Enzymol., 194:3-21). Yeast transformations were performed using standardprotocols (Gietz et al., 1992, Nucleic Acids Res., 20:1425. Ito et al.,1983, J. Bacteriol., 153:163-168). Plasmid DNAs were isolated from yeaststrains by a standard method (Hoffman and Winston, 1987, Gene,57:267-272).

11.1.2. Plasmid and Yeast Strain Construction

The coding region of human fchd540 was amplified by PCR and cloned inframe into pGBT9 (Bartel et al., 1993, Cellular Interactions inDevelopment. pp. 153-159) resulting in plasmid pGBT9-fchd540.pGBT9-fchd540 was transformed into two-hybrid screening strain HF7c andone resulting transformant was designated TB35.

11.1.3. Two-Hybrid Screening

Two-hybrid screening was carried out essentially as described (Bartel etal., 1993, supra) using TB35 as the recipient strain and a human breasttwo-hybrid library.

11.1.4. Paper Filter Beta-Galactosidase Assays

The paper filter beta-galactosidase (beta-gal) assay was performedessentially as previously described (Brill et al., 1994, Mol. Biol. Cell5: 297-312).

11.2. Results

11.2.1. Strong Physical Interaction of rchd534 and fchd540 Measured byTwo-hybrid Assay

The fchd540 coding sequence was amplified by PCR and cloned into pGBT9creating a GAL4 DNA-binding domain-fchd540 fusion gene. The screeningstrain HF7c was transformed with this construct. The rchd534 codingsequence was cloned into pGAD424 (Bartel et al., 1993, supra) creating aGAL4 transcriptional activation domain-rchd534 fusion gene, which wasthen used to transform strain Y187.

Yeast expression plasmids encoding the GAL4 DNA-binding domain eitheralone or fused in frame to fchd540, rchd534, Drosophilia MAD, DPC4, orp53 were transformed into MATa two-hybrid screening strain HF7c. Yeastexpression plasmids encoding the GAL4 transcriptional activation domainalone and GAL4 activation domain fusions to rchd534 and SV40 weretransformed into MATA two-hybrid screening strain Y187. p53 and SV40interact with each other and should not interact with the experimentalproteins. The HF7c transformants were propagated as stripes on semisolidsynthetic complete medium lacking L-tryptophan and the Y187transformants were grown as stripes on semisolid synthetic completemedium lacking L-leucine. Both sets of stripes were replica plated inthe form of a grid onto a single rich YPAD plate and the haploid strainsof opposite mating types were allowed to mate overnight at 30° C. Theyeast strains on the mating plate were then replica plated to asynthetic complete plate lacking L-leucine and L-tryptophan to selectfor diploids and incubated at 30° C. overnight. Diploid strains on thesynthetic complete plate lacking L-leucine and L-tryptophan were replicaplated to a synthetic complete plate lacking L-leucine, L-tryptophan,and L-histidine to assay HIS-3 expression and a paper filter on asynthetic complete plate lacking L-leucine and L-tryptophan. The nextday the paper filter was subjected to the paper filterbeta-galactosidase assay to measure expression of the lacZ reportergene. HIS3 expression was scored after 3 days of growth at 30° C. Theresults are shown in Table 3.

The rchd534 fish protein was found to interact strongly with the fchd540bait protein and not to interact with the rchd534, MAD, DPC4, p53, andGAL4 DNA binding domain bait proteins. This result demonstrated thatrchd534 and fchd540 strongly physically interact with each other withsignificant specificity.

11.2.2. Identification of Proteins that Physically Interact with fchd540

The fchd540 coding sequence was amplified by PCR and cloned into pGBT9(Bartel et al., 1993, supra) creating a GAL4 DNA-binding domain-fchd540fusion gene. HF7c was transformed with this construct resulting instrain TB35. TB35 grew on synthetic complete medium lacking L-tryptophanbut not on synthetic complete medium lacking L-tryptophan andL-histidine demonstrating that the GAL4 DNA-binding domain-fchd540fusion does not have intrinsic transcriptional activation activity.

TB35 was transformed with the human breast two-hybrid library and 5million transformants were obtained. The transformants were plated onsynthetic complete medium lacking L-leucine, L-tryptophan , andL-histidine and yeast colonies that both grew on synthetic completemedium lacking L-leucine, L-tryptophan, and L-histidine and expressedthe beta-galactosidase reporter gene were identified. The 30 strainswith the strongest beta-galactosidase induction were characterized.Library plasmids were isolated from these strains, and the 5' ends ofall of the cDNA inserts were sequenced.

11.2.3. Retransformation and Specificity Testing of tchv03A and tchvR4A

Two of the plasmids that encoded the strongest interactors were found tocontain rchd534 cDNAs. Plasmid tchv03A was found to encode amino acids17-235 of rchd534 and plasmid tchvR4A was found to encode amino acids25-235 of rchd534.

It was confirmed that these rchd534 cDNAs encode proteins thatphysically interact specifically with fchd540. Yeast expression plasmidsencoding the GAL4 DNA-binding domain either alone or fused in frame tofchd540, rchd534, Drosophila MAD, DPC4, and p53 were transformed intoMATa two-hybrid screening strain HF7c. Yeast expression plasmidsencoding the GAL4 transcriptional activation domain (GAL4 AD) alone andGAL4 activation domain fusions to tchv03a, tchvR4A and SV40 weretransformed into MATα two-hybrid screening strain Y187. p53 and SV40interact with each other and should not interact with the experimentalproteins. The HF7c transformants were propagated as stripes onsemi-solid synthetic complete medium lacking L-leucine. Both sets ofstripes were replica plated in the form of a grid onto a single richYPAD plate and the haploid strains of opposite mating types were allowedto mate overnight at 30° C. The yeast strains on the mating plate werethen replica plated to a synthetic complete plate lacking L-leucine andL-tryptophan to select for diploids and incubated at 30° C. overnight.Diploid strains on the synthetic complete plate lacking L-leucine andL-tryptophan were replica plated to a synthetic complete plate lackingL-leucine, L-tryptophan, and L-histidine to assay HIS3 expression and apaper filter on a synthetic complete plate lacking L-leucine andL-tryptophan. The next day the paper filter was subjected to the paperfilter beta-galactosidase assay to measure expression of the lacZreporter gene. HIS3 expression was scored after 3 days of growth at 30°C. The results are shown in the table below. The strength or absence ofphysical interaction between each combination of test proteins islisted. Strong interactions are defined as interactions that cause theactivation of both the HIS3 and lacZ reporter genes.

                  TABLE 3                                                         ______________________________________                                        cDNA-GAL4 Activation Domain Fusion                                              Tested                                                                                                              GAL4 AD                                 rchd534 tchv03A tchvR4A SV40 alone                                          ______________________________________                                        GAL4 DNA-                                                                       Binding                                                                       Domain                                                                        Fusions                                                                       fchd540 Strong Strong Strong None None                                        rchd534 None None None None None                                              Dros. MAD None None None None None                                            DPC4 None None None None None                                                 p53 None None None Strong None                                                GAL4 DNA- None None None None None                                            Binding                                                                       Domain                                                                        alone                                                                       ______________________________________                                    

The tchv03A and tchvR4A fish proteins were found to interact stronglywith the fchd540 bait protein and to not interact with the rchd534, MAD,DPC4, p53, and GAL4 DNA binding domain bait proteins. These resultsconfirm the result that the rchd534 and fchd540 proteins interactstrongly with each other.

11.3. Further Analysis of rchd534 and fchd540 Function

The significance of the rchd534/fchd540 protein interaction wasconfirmed by examination of their expression and activity in human cellsand animal models.

11.3.1 Tissue Expression Patterns

The expression patterns were examined using in situ hybridizationtechniques. Fluorescently labeled DNA probes of both the rchd534 andfchd540 genes were used to probe human carotid endartectomy samples. Theexpression of rchd534 and fchd540 was specific to endothelial cellslining the luminal surface of the carotid artery. Neither gene showedexpression in any other cell type present in the arterial tissue sample,including smooth muscle cells and macrophages. Thus, the specificity oftheir expression in a cell-type that is found only in vascular tissue,including atherosclerotic plaques, in addition to their up-regulationunder the shear stress cardiovascular disease paradigm, indicate thatrchd534 and fchd540 are excellent and specific targets for therapeuticintervention.

11.3.2. Cellular Localization

The cellular localization of the rchd534 and fchd540 proteins in bovineaortic endothelial cells (BAECs) was examined in relationship to otherproteins involved in the TGF-β signalling pathway. In all experiments,the rchd534 and fchd540 proteins were located in the cytoplasm MADR2 waslocated in the cytoplasm when transfected alone and in the nucleus whenco-transfected with activated TβRI or when TGF-β was added to theculture medium. Co-transfection of rchd534 or fchd540 with MADR2prevented the localization of MADR2 in the nucleus in response to TGF-βsignalling.

11.3.3. Protein Interactions in Human Cells

The interaction of the rchd534 and fchd540 proteins, observed in yeastcells as described above, was tested in mammalian endothelial celltissue culture. Either bovine aortic endothelial cells (BAECs) or 293cells (human embryonic kidney cells, ATCC Accession No. CRL-1573) weretransfected with constructs encoding both the rchd534 and fchd540proteins, each fused to a different flag peptide allowing for specificimmunoprecipitation. The rchd534 and fchd540 proteins were found toco-immunoprecipitate as heterodimers in extracts produced from both 293cells and BAECs. The co-immunoprecipitation of rchd534 and fchd540further supports that these proteins interact in human cells that arephysiologically relevant to cardiovascular disease.

The ability of the rchd534 and fchd540 proteins to interact withthemselves and with other protein members of the TGF-β signallingpathway (MADR1, MADR2, DPC4, TbR1, TSR1, ActR1b, ALK3, ALK6), was testedusing this co-immunoprecipitation method. Each gene was transfectedalone and in various combinations with other TGF-β pathway genes ineither 293 cells or BAECs. The rchd534 protein formed homodimers in 293cells and BAECS. The fchd540 protein did not form homodimers in 293cells or BAECs. As mentioned above, the rchd534 and fchd540 proteinsformed heterodimers in 293 cells and BAECs. This interaction is about 50fold stronger in BAECs than 293 cells based on equal amounts of protein.However, the rchd534-fchd540 protein interaction was significantly lessavid than the rchd534 protein's interaction with itself.

The rchd534 protein interacted with MADR1, MADR2, and DPC4 in 293 cellsand BAECs. The strength of MADR1 and MADR2 interactions was about thesame between 293 cells and BAECs and much greater in BAECs for DPC4. Thefchd540 protein interacted very weakly with MADR1, ADR2, and DPC4 in 293cells. The rchd534 protein interacted strongly with activated forms ofTβRI and ActRI and weakly with activated ALK6 in 293 cells. The fchd540protein interacted strongly with activated TβRI and ALK6 receptors, andweakly with activated forms of TSRI, ALK3, and ActRIb in 293 cells.Thus, in addition to the interaction of the rchd534 and fchd540proteins, the interaction of the rchd534 protein with itself, as well asthe interaction of the rchd534 protein and the fchd540 protein with theother proteins in the TGF-β pathway described above are excellenttargets for therapeutic intervention.

11.3.4. Effect of Expression on TGF-β Signalling

The effect of both rchd534 and fchd540 on the TGF-β signalling pathwaywas tested in vitro. Primary BAECs were transfected with a constructcalled p3TP-Lux, containing a TGF-β responsive promoter fused to areporter gene (Wrana et al., 1994, Nature 370: 341-347). The rchd534gene or the fchd540 gene in pCI expression vectors (Promega) wastransfected with and without MADR1 (pCMV5MADR1-Flag, Hoodless et al.1996 Cell 85: 489-500) or MADR2 (pCMV5MADR2-Flag, Eppert et al. 1996Cell 86: 543-552). The TGF-β response was induced 20-fold by eitherMADR1 or MADR2. Co-expression of either rchd534 or fchd540 completelyeliminated this induction. Thus, the rchd534 and fchd540 proteinsinhibited MADR1- and MADR2-mediated TGF-β signalling in endothelialcells. These results further demonstrate that the interactions of eitherthe rchd534 protein or the fchd540 protein with MADR2 or with activatedTβR1 are excellent targets for therapeutic intervention. As describedabove, the expression of rchd534 and fchd540 is specific, withinarterial tissue, to endothelial cells. Accordingly, the rchd534 andcchd540 genes may be targets for intervention in a variety ofinflammatory and fibroproliferative disorders that involve endothelialcells, including, but not limited to, cancer angiogenesis, inflammation,and fibrosis.

12. EXAMPLE: ANTISENSE AND RIBOZYME MOLECULES FOR INHIBITION OF RCHD534AND FCHD540 EXPRESSION

The principles presented in Section 5.6.1.1, above, can be used todesign oligonucleotides for use in inhibiting the expression of targetgenes, such as the rchd534 or fchd540 genes.

The following antisense molecules can be used to inhibit the expressionof the rchd534 gene.

Antisense

a) 5'-CATTTCATTTCATACAA-3' (SEQ ID NO:20) which is complementary tonucleotides -14 to +3 of rchd534 in FIGS. 6A-6C.

b) 5'-CATTTCATTTCATACAATATATG-3' (SEQ ID NO:21) which is complementaryto nucleotides -20 to +3 of rchd534 in FIGS. 6A-6C.

c) 5'-CATTTCATTTCATACAATATATGGCCTTT-3' (SEQ ID NO:22) which iscomplementary to nucleotides -26 to +3 of rchd534 in FIGS. 6A-6C.

d) 5'-CATTTCATTTCATACAATATATGGCCTTTTGTGGC-3' (SEQ ID NO:23) which iscomplementary to nucleotides -32 to +3 of rchd534 in FIGS. 6A-6C.

e) 5'-GGACATTTCATTTCATACAATATATGGCCTTTTGT3' (SEQ ID NO:24) which iscomplementary to nucleotides -29 to +6 of rchd534 in FIGS. 6A-6C.

f) 5'-TTCATTTCATACAATATATGGCCTTTTGT-3' (SEQ ID NO:25) which iscomplementary to nucleotides -29 to -1 of rchd534 in FIGS. 6A-6C.

g) 5'-TCATACAATATATGGCCTTTTGT-3' (SEQ ID NO:26) which is complementaryto nucleotides -29 to -7 of rchd534 in FIGS. 6A-6C.

h) 5'-AATATATGGCCTTTTGT-3' (SEQ ID NO:27) which is complementary tonucleotides -29 to -13 of rchd534 in FIGS. 6A-6C.

The following antisense molecules can be used to inhibit the expressionof the fchd540 gene:

a) 5'-CATGCGGGGCGAGGAGG-3' (SEQ ID NO:28) which is complementary tonucleotides -14 to +3 of fchd540 in FIGS. 2A-2C.

b) 5'-CATGCGGGGCGAGGAGGCGAGGA-3' (SEQ ID NO:29) which is complementaryto nucleotides -20 to +3 of fchd540 in FIGS. 2A-2C.

c) 5'-CATGCGGGGCGAGGAGGCGAGGAGAAAAG-3' (SEQ ID NO:30) which iscomplementary to nucleotides -26 to +3 of fchd540 in FIGS. 2A-2C.

d) 5'-CATGCGGGGCGAGGAGGCGAGGAGAAAAGTCGTTT-3' (SEQ ID NO:31) which iscomplementary to nucleotides -32 to +3 of fchd540 in FIGS. 2A-2C.

e) 5'-GAACATGCGGGGCGAGGAGGCGAGGAGAAAAGTCG-3' (SEQ ID NO:32) which iscomplementary to nucleotides -29 to +6 of fchd540 in FIGS. 2A-2C.

f) 5'-GCGGGGCGAGGAGGCGAGGAGAAAAGTCG-3' (SEQ ID NO:33) which iscomplementary to nucleotides -29 to -1 of fchd540 in FIGS. 2A-2C.

g) 5'-CGAGGAGGCGAGGAGAAAAGTCG-3'(SEQ ID NO:34) which is complementary tonucleotides -29 to -7 of fchd540 in FIGS. 2A-2C.

h) 5'-GGCGAGGAGAAAAGTCG-3' (SEQ ID NO:35) which is complementary tonucleotides -29 to -13 of fchd540 in FIGS. 2A-2C.

Ribozymes

The central, catalytic portion of a hammerhead ribozyme molecule consistof the following sequence:

5'-CAAAGCNGNXXXXNCNGAGNAGUC-3' (SEQ ID NO:36);

wherein the 5'-proximal CA bases hybridize to a complementary 5'-UG-3'in the target mRNA. The first four underlined bases form a stem by basepairing with the second set of underlined bases, with the interveningbases, shown as X's, forming a non-pairing loop. In order to hybridizeto a target mRNA, a hammerhead ribozyme contains additional basesflanking each end of the central segment shown above. The 5' ribozymeflanking segment is complementary to the respective flanking sequencesimmediately 3' to the target UG; and the 3' flanking segment iscomplementary to the respective flanking sequence beginning two basesupstream of the target U, and extending 5'-ward (in effect, skipping thefirst base upstream of the target U). Cleavage occurs between first andsecond bases upstream of (i.e., 5' to) the U in the target 5'-UG-3'site.

The following ribozyme molecules can be used to inhibit the expressionof the rchd534 gene:

a) 5'-GGUGGAGCCCCAGGGCAUUACCUCAAAGCNGNXXXXNCNGAGNAGUCGUGGGCAAGGUGGGCACUCAGGUGGG-3' (SEQ ID NO:37) which will cleave the rchd534mRNA between nucleotides 716 and 717 in FIGS. 6A-6C.

b) 5'-GUGUCUCUAUGGGUUUGCCCAAAGCNGNXXXXNCNGAGNAGUCUCUGGACAUUUCAUUUCAUAC-3' (SEQ ID NO:38) which will cleave the rchd534 mRNAbetween nucleotides 1040 and 1041 in FIGS. 6A-6C.

c) 5'-GGCCCUCUCGCCGUCGGGCUCCUUGCUGAGCAAAGCNGNXXXXNCNGAGNAGUCGAUGCCGAAGCCGAUCUUGCUGCGCG-3' (SEQ ID NO:39) which will cleavebetween nucleotides 1421 and 1422 in FIGS. 6A-6C.

The following ribozyme molecules can be used to inhibit the expressionof the fchd540 gene:

a) 5'-CGUUUGCCUGCUAAGGAGCGAACAAAGCNGNXXXXNCNGAGNAGUCGAUGUUUCUUUGUGAGUCGGGCGCCG-3'(SEQ ID NO:40), which will cleave the fchd540mRNA between nucleotides -53 and -52 in FIGS. 2A-2C.

b) 5'-CGCCGGACGAGCGCAGAUCGUUUGGUCCUGAACAAAGCNGNXXXXNCNGAGNAGUCCGGGGCGAGGAGGCGAGGAGAAAAGUCG-3'(SEQ ID NO:41), which will cleavethe fchd540 mRNA between nucleotides -1 and +1 in FIGS. 2A-2C.

c) 5'-GGAGUAAGGAGGGGGGGGAGACUCUAGUUCGCAAAGCNGNXXXXNCNGAGNAGUCAGUCGGCUAAGGUGAUGGGGGUUGCAGCACACC-3' (SEQ ID NO:42) which willcleave the fchd540 mRNA between nucleotides +602 and +603 in FIGS.2A-2C.

13. DEPOSIT OF MICROORGANISMS

The following microorganisms were deposited with the American TypeCulture Collection (ATCC), Rockville, Md., on the indicated dates andassigned the indicated accession numbers:

    ______________________________________                                        Microorganism                                                                            ATCC Accession No.                                                                             Date of Deposit                                   ______________________________________                                        pFCHD531   69983            February 7, 1996                                    pFCHD540 69984 February 7, 1996                                               fchd545 69974 January 5, 1996                                               ______________________________________                                    

The following microorganism was deposited with the Agricultural ResearchService Culture Collection (NRRL), Peoria, Ill., on the indicated dateand assigned the indicated accession number:

    ______________________________________                                        Microorganism                                                                             NRRL Accession No.                                                                          Date of Deposit                                     ______________________________________                                        FCHD534     B-21459       June 6, 1995                                        ______________________________________                                    

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 44                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1953 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Coding Se - #quence                                             (B) LOCATION: 162...1871                                                      (D) OTHER INFORMATION:                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  GGCACGAGTC GGAGCCGGGC GGAGGGGAGG GGGGAAAGAG GAGCGCAGGG - #TGAGAGTG    AG    60                                                                        - -  CCGCAGGCTT CGGGAGGCGA GGGGGCGGGG GGAGCAGCGC CGAGGYCGCC - #GCCTCCGCC    T   120                                                                         - -  CCGCCGCCTA GGACTAGGGG GTGGGGGACG GACAAGCCCC G ATG - #CCG GGG GAG       ACG   176                                                                                         - #                  - #           Met Pro Gly Glu -      #Thr                                                                                              - #                  - #            1      - #             5                                                                              - -  GAA GAG CCG AGA CCC CCG GAG CAG CAG GAC - #CAG GAA GGG GGA GAG GCG         224                                                                         Glu Glu Pro Arg Pro Pro Glu Gln Gln Asp - #Gln Glu Gly Gly Glu Ala                            10 - #                 15 - #                 20              - -  GCC AAG GCG GCT CCG GAG GAG CCC CAA CAA - #CGG CCC CCT GAG GCG GTC         272                                                                         Ala Lys Ala Ala Pro Glu Glu Pro Gln Gln - #Arg Pro Pro Glu Ala Val                        25     - #             30     - #             35                  - -  GCG GCG GCG CCT GCA GGG ACC ACT AGC AGC - #CGC GTG CTG AGG GGA GGT         320                                                                         Ala Ala Ala Pro Ala Gly Thr Thr Ser Ser - #Arg Val Leu Arg Gly Gly                    40         - #         45         - #         50                      - -  CGG GAC CGA GGC CGG GCC GCT GCG GCC GCC - #GCC GCC GCA GCT GTG TCC         368                                                                         Arg Asp Arg Gly Arg Ala Ala Ala Ala Ala - #Ala Ala Ala Ala Val Ser                55             - #     60             - #     65                          - -  CGC CGG AGG AAG GCC GAG TAT CCC CGC CGG - #CGG AGG AGC AGC CCC AGC         416                                                                         Arg Arg Arg Lys Ala Glu Tyr Pro Arg Arg - #Arg Arg Ser Ser Pro Ser            70                 - # 75                 - # 80                 - # 85       - -  GCC AGG CCT CCC GAC GTC CCC GGG CAG CAG - #CCC CAG GCC GCG AAG TCC         464                                                                         Ala Arg Pro Pro Asp Val Pro Gly Gln Gln - #Pro Gln Ala Ala Lys Ser                            90 - #                 95 - #                 100             - -  CCG TCT CCA GTT CAG GGC AAG AAG AGT CCG - #CGA CTC CTA TGC ATA GAA         512                                                                         Pro Ser Pro Val Gln Gly Lys Lys Ser Pro - #Arg Leu Leu Cys Ile Glu                        105     - #            110     - #            115                 - -  AAA GTA ACA ACT GAT AAA GAT CCC AAG GAA - #GAA AAA GAG GAA GAA GAC         560                                                                         Lys Val Thr Thr Asp Lys Asp Pro Lys Glu - #Glu Lys Glu Glu Glu Asp                    120         - #        125         - #        130                     - -  GAT TCT GCC CTC CCT CAG GAA GTT TCC ATT - #GCT GCA TCT AGA CCT AGC         608                                                                         Asp Ser Ala Leu Pro Gln Glu Val Ser Ile - #Ala Ala Ser Arg Pro Ser                135             - #    140             - #    145                         - -  CGG GGC TGG CGT AGT AGT AGG ACA TCT GTT - #TCT CGC CAT CGT GAT ACA         656                                                                         Arg Gly Trp Arg Ser Ser Arg Thr Ser Val - #Ser Arg His Arg Asp Thr            150                 - #155                 - #160                 -         #165                                                                             - -  GAG AAC ACC CGA AGC TCT CGG TCC AAG ACC - #GGT TCA TTG CAG CTC        ATT     704                                                                      Glu Asn Thr Arg Ser Ser Arg Ser Lys Thr - #Gly Ser Leu Gln Leu Ile                           170 - #                175 - #                180             - -  TGC AAG TCA GAA CCA AAT ACA GAC CAA CTT - #GAT TAT GAT GTT GGA GAA         752                                                                         Cys Lys Ser Glu Pro Asn Thr Asp Gln Leu - #Asp Tyr Asp Val Gly Glu                        185     - #            190     - #            195                 - -  GAG CAT CAG TCT CCA GGT GGC ATT AGT GGT - #GAA GAG GAA GAG GAG GAG         800                                                                         Glu His Gln Ser Pro Gly Gly Ile Ser Gly - #Glu Glu Glu Glu Glu Glu                    200         - #        205         - #        210                     - -  GAA GAA GAG ATG TTA ATC AGT GAA GAG GAG - #ATA CCA TTC AAA GAT GAT         848                                                                         Glu Glu Glu Met Leu Ile Ser Glu Glu Glu - #Ile Pro Phe Lys Asp Asp                215             - #    220             - #    225                         - -  CCA AGA GAT GAG ACC TAC AAA CCC CAC TTA - #GAA AGG GAA ACC CCA AAG         896                                                                         Pro Arg Asp Glu Thr Tyr Lys Pro His Leu - #Glu Arg Glu Thr Pro Lys            230                 - #235                 - #240                 -         #245                                                                             - -  CCA CGG AGA AAA TCA GGG AAG GTA AAA GAA - #GAG AAG GAG AAG AAG        GAA     944                                                                      Pro Arg Arg Lys Ser Gly Lys Val Lys Glu - #Glu Lys Glu Lys Lys Glu                           250 - #                255 - #                260             - -  ATT AAA GTG GAA GTA GAG GTG GAG GTG AAA - #GAA GAG GAG AAT GAA ATT         992                                                                         Ile Lys Val Glu Val Glu Val Glu Val Lys - #Glu Glu Glu Asn Glu Ile                        265     - #            270     - #            275                 - -  AGA GAG GAT GAG GAA CCT CCA AGG AAG AGA - #GGA AGA AGA CGA AAA GAT        1040                                                                         Arg Glu Asp Glu Glu Pro Pro Arg Lys Arg - #Gly Arg Arg Arg Lys Asp                    280         - #        285         - #        290                     - -  GAC AAA AGT CCA CGT TTA CCC AAA AGG AGA - #AAA AAG CCT CCA ATC CAG        1088                                                                         Asp Lys Ser Pro Arg Leu Pro Lys Arg Arg - #Lys Lys Pro Pro Ile Gln                295             - #    300             - #    305                         - -  TAT GTC CGT TGT GAG ATG GAA GGA TGT GGA - #ACT GTC CTT GCC CAT CCT        1136                                                                         Tyr Val Arg Cys Glu Met Glu Gly Cys Gly - #Thr Val Leu Ala His Pro            310                 - #315                 - #320                 -         #325                                                                             - -  CGC TAT TTG CAG CAC CAC ATT AAA TAC CAG - #CAT TTG CTG AAG AAG        AAA    1184                                                                      Arg Tyr Leu Gln His His Ile Lys Tyr Gln - #His Leu Leu Lys Lys Lys                           330 - #                335 - #                340             - -  TAT GTA TGT CCC CAT CCC TCC TGT GGA CGA - #CTC TTC AGG CTT CAG AAG        1232                                                                         Tyr Val Cys Pro His Pro Ser Cys Gly Arg - #Leu Phe Arg Leu Gln Lys                        345     - #            350     - #            355                 - -  CAA CTT CTG CGA CAT GCC AAA CAT CAT ACA - #GAT CAA AGG GAT TAT ATC        1280                                                                         Gln Leu Leu Arg His Ala Lys His His Thr - #Asp Gln Arg Asp Tyr Ile                    360         - #        365         - #        370                     - -  TGT GAA TAT TGT GCT CGG GCC TTC AAG AGT - #TCC CAC AAT CTG GCA GTG        1328                                                                         Cys Glu Tyr Cys Ala Arg Ala Phe Lys Ser - #Ser His Asn Leu Ala Val                375             - #    380             - #    385                         - -  CAC CGG ATG ATT CAC ACT GGC GAG AAG CCA - #TTA CAA TGT GAG ATC TGT        1376                                                                         His Arg Met Ile His Thr Gly Glu Lys Pro - #Leu Gln Cys Glu Ile Cys            390                 - #395                 - #400                 -         #405                                                                             - -  GGA TTT ACT TGT CGA CAA AAG GCA TCT CTT - #AAT TGG CAC ATG AAG        AAA    1424                                                                      Gly Phe Thr Cys Arg Gln Lys Ala Ser Leu - #Asn Trp His Met Lys Lys                           410 - #                415 - #                420             - -  CAT GAT GCA GAC TCC TTC TAC CAG TTT TCT - #TGC AAT ATC TGT GGC AAA        1472                                                                         His Asp Ala Asp Ser Phe Tyr Gln Phe Ser - #Cys Asn Ile Cys Gly Lys                        425     - #            430     - #            435                 - -  AAA TTT GAG AAG AAG GAC AGC GTA GTG GCA - #CAC AAG GCA AAA AGC CAC        1520                                                                         Lys Phe Glu Lys Lys Asp Ser Val Val Ala - #His Lys Ala Lys Ser His                    440         - #        445         - #        450                     - -  CCT GAG GTG CTG ATT GCA GAA GCT CTG GCT - #GCC AAT GCA GGC GCC CTC        1568                                                                         Pro Glu Val Leu Ile Ala Glu Ala Leu Ala - #Ala Asn Ala Gly Ala Leu                455             - #    460             - #    465                         - -  ATC ACC AGC ACA GAT ATC TTG GGC ACT AAC - #CCA GAG TCC CTG ACG CAG        1616                                                                         Ile Thr Ser Thr Asp Ile Leu Gly Thr Asn - #Pro Glu Ser Leu Thr Gln            470                 - #475                 - #480                 -         #485                                                                             - -  CCT TCA GAT GGT CAG GGT CTT CCT CTT CTT - #CCT GAG CCC TTG GGA        AAC    1664                                                                      Pro Ser Asp Gly Gln Gly Leu Pro Leu Leu - #Pro Glu Pro Leu Gly Asn                           490 - #                495 - #                500             - -  TCA ACC TCT GGA GAG TGC CTA CTG TTA GAA - #GCT GAA GGG ATG TCA AAG        1712                                                                         Ser Thr Ser Gly Glu Cys Leu Leu Leu Glu - #Ala Glu Gly Met Ser Lys                        505     - #            510     - #            515                 - -  TCA TAC TGC AGT GGG ACG GAA CGG GTG AGC - #CTG ATG GCT GAT GGG AAG        1760                                                                         Ser Tyr Cys Ser Gly Thr Glu Arg Val Ser - #Leu Met Ala Asp Gly Lys                    520         - #        525         - #        530                     - -  ATC TTT GTG GGA AGC GGC AGC AGT GGA GGC - #ACT GAA GGG CTG GTT ATG        1808                                                                         Ile Phe Val Gly Ser Gly Ser Ser Gly Gly - #Thr Glu Gly Leu Val Met                535             - #    540             - #    545                         - -  AAC TCA GAT ATA CTC GGT GCT ACC ACA GAG - #GTT CTG ATT GAA GAT TCA        1856                                                                         Asn Ser Asp Ile Leu Gly Ala Thr Thr Glu - #Val Leu Ile Glu Asp Ser            550                 - #555                 - #560                 -         #565                                                                             - -  GAC TCT GCC GGA CCT TAGTGGACAG GAAGACTTGG GGCAT - #GGGAC AGCTCAGAC    T T  1912                                                                       Asp Ser Ala Gly Pro                                                                           570                                                           - -  TGTATTTAAA AGTTAAAAAG GACAAAAAAA AAAAAAAAAA A   - #                      - # 1953                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 570 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  Met Pro Gly Glu Thr Glu Glu Pro Arg Pro - #Pro Glu Gln Gln Asp Gln        1               5 - #                 10 - #                 15              - -  Glu Gly Gly Glu Ala Ala Lys Ala Ala Pro - #Glu Glu Pro Gln Gln Arg                   20     - #             25     - #             30                  - -  Pro Pro Glu Ala Val Ala Ala Ala Pro Ala - #Gly Thr Thr Ser Ser Arg               35         - #         40         - #         45                      - -  Val Leu Arg Gly Gly Arg Asp Arg Gly Arg - #Ala Ala Ala Ala Ala Ala           50             - #     55             - #     60                          - -  Ala Ala Ala Val Ser Arg Arg Arg Lys Ala - #Glu Tyr Pro Arg Arg Arg       65                 - # 70                 - # 75                 - # 80       - -  Arg Ser Ser Pro Ser Ala Arg Pro Pro Asp - #Val Pro Gly Gln Gln Pro                       85 - #                 90 - #                 95              - -  Gln Ala Ala Lys Ser Pro Ser Pro Val Gln - #Gly Lys Lys Ser Pro Arg                   100     - #            105     - #            110                 - -  Leu Leu Cys Ile Glu Lys Val Thr Thr Asp - #Lys Asp Pro Lys Glu Glu               115         - #        120         - #        125                     - -  Lys Glu Glu Glu Asp Asp Ser Ala Leu Pro - #Gln Glu Val Ser Ile Ala           130             - #    135             - #    140                         - -  Ala Ser Arg Pro Ser Arg Gly Trp Arg Ser - #Ser Arg Thr Ser Val Ser       145                 - #150                 - #155                 -         #160                                                                             - -  Arg His Arg Asp Thr Glu Asn Thr Arg Ser - #Ser Arg Ser Lys Thr        Gly                                                                                              165 - #                170 - #                175            - -  Ser Leu Gln Leu Ile Cys Lys Ser Glu Pro - #Asn Thr Asp Gln Leu Asp                   180     - #            185     - #            190                 - -  Tyr Asp Val Gly Glu Glu His Gln Ser Pro - #Gly Gly Ile Ser Gly Glu               195         - #        200         - #        205                     - -  Glu Glu Glu Glu Glu Glu Glu Glu Met Leu - #Ile Ser Glu Glu Glu Ile           210             - #    215             - #    220                         - -  Pro Phe Lys Asp Asp Pro Arg Asp Glu Thr - #Tyr Lys Pro His Leu Glu       225                 - #230                 - #235                 -         #240                                                                             - -  Arg Glu Thr Pro Lys Pro Arg Arg Lys Ser - #Gly Lys Val Lys Glu        Glu                                                                                              245 - #                250 - #                255            - -  Lys Glu Lys Lys Glu Ile Lys Val Glu Val - #Glu Val Glu Val Lys Glu                   260     - #            265     - #            270                 - -  Glu Glu Asn Glu Ile Arg Glu Asp Glu Glu - #Pro Pro Arg Lys Arg Gly               275         - #        280         - #        285                     - -  Arg Arg Arg Lys Asp Asp Lys Ser Pro Arg - #Leu Pro Lys Arg Arg Lys           290             - #    295             - #    300                         - -  Lys Pro Pro Ile Gln Tyr Val Arg Cys Glu - #Met Glu Gly Cys Gly Thr       305                 - #310                 - #315                 -         #320                                                                             - -  Val Leu Ala His Pro Arg Tyr Leu Gln His - #His Ile Lys Tyr Gln        His                                                                                              325 - #                330 - #                335            - -  Leu Leu Lys Lys Lys Tyr Val Cys Pro His - #Pro Ser Cys Gly Arg Leu                   340     - #            345     - #            350                 - -  Phe Arg Leu Gln Lys Gln Leu Leu Arg His - #Ala Lys His His Thr Asp               355         - #        360         - #        365                     - -  Gln Arg Asp Tyr Ile Cys Glu Tyr Cys Ala - #Arg Ala Phe Lys Ser Ser           370             - #    375             - #    380                         - -  His Asn Leu Ala Val His Arg Met Ile His - #Thr Gly Glu Lys Pro Leu       385                 - #390                 - #395                 -         #400                                                                             - -  Gln Cys Glu Ile Cys Gly Phe Thr Cys Arg - #Gln Lys Ala Ser Leu        Asn                                                                                              405 - #                410 - #                415            - -  Trp His Met Lys Lys His Asp Ala Asp Ser - #Phe Tyr Gln Phe Ser Cys                   420     - #            425     - #            430                 - -  Asn Ile Cys Gly Lys Lys Phe Glu Lys Lys - #Asp Ser Val Val Ala His               435         - #        440         - #        445                     - -  Lys Ala Lys Ser His Pro Glu Val Leu Ile - #Ala Glu Ala Leu Ala Ala           450             - #    455             - #    460                         - -  Asn Ala Gly Ala Leu Ile Thr Ser Thr Asp - #Ile Leu Gly Thr Asn Pro       465                 - #470                 - #475                 -         #480                                                                             - -  Glu Ser Leu Thr Gln Pro Ser Asp Gly Gln - #Gly Leu Pro Leu Leu        Pro                                                                                              485 - #                490 - #                495            - -  Glu Pro Leu Gly Asn Ser Thr Ser Gly Glu - #Cys Leu Leu Leu Glu Ala                   500     - #            505     - #            510                 - -  Glu Gly Met Ser Lys Ser Tyr Cys Ser Gly - #Thr Glu Arg Val Ser Leu               515         - #        520         - #        525                     - -  Met Ala Asp Gly Lys Ile Phe Val Gly Ser - #Gly Ser Ser Gly Gly Thr           530             - #    535             - #    540                         - -  Glu Gly Leu Val Met Asn Ser Asp Ile Leu - #Gly Ala Thr Thr Glu Val       545                 - #550                 - #555                 -         #560                                                                             - -  Leu Ile Glu Asp Ser Asp Ser Ala Gly Pro                                                  565 - #                570                                    - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3103 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Coding Se - #quence                                             (B) LOCATION: 288...1565                                                      (D) OTHER INFORMATION:                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  GCACGAGCGG AGAGCCGCGC AGGGCGCGGG CCGCGCGGGG TGGGGCAGCC - #GGAGCGCA    GG    60                                                                        - -  CCCCCGATCC CCGGCGGGCG CCCCCGGGCC CCCGCGCGCG CCCCGGCCTC - #CGGGAGACT    G   120                                                                         - -  GCGCATGCCA CGGAGCGCCC CTCGGGCCGC CGCCGCTCCT GCCCGGGCCC - #CTGCTGCTG    C   180                                                                         - -  TGCTGTCGCC TGCGCCTGCT GCCCCAACTC GGCGCCCGAC TCACAAAGAA - #ACATCATGT    T   240                                                                         - -  CGCTCCTTAG CAGGCAAACG ACTTTTCTCC TCGCCTCCTC GCCCCGC A - #TG TTC        AGG     296                                                                                       - #                  - #                 Met - # Phe      Arg                                                                                               - #                  - #                  - #1              - -  ACC AAA CGA TCT GCG CTC GTC CGG CGT CTC - #TGG AGG AGC CGT GCG CCC         344                                                                         Thr Lys Arg Ser Ala Leu Val Arg Arg Leu - #Trp Arg Ser Arg Ala Pro                 5            - #      10            - #      15                          - -  GGC GGC GAG GAC GAG GAG GAG GGC GCA GGG - #GGA GGT GGA GGA GGA GGC         392                                                                         Gly Gly Glu Asp Glu Glu Glu Gly Ala Gly - #Gly Gly Gly Gly Gly Gly            20                 - # 25                 - # 30                 - # 35       - -  GAG CTG CGG GGA GAA GGG GCG ACG GAC AGC - #CGA GCG CAT GGG GCC GGT         440                                                                         Glu Leu Arg Gly Glu Gly Ala Thr Asp Ser - #Arg Ala His Gly Ala Gly                            40 - #                 45 - #                 50              - -  GGC GGC GGC CCG GGC AGG GCT GGA TGC TGC - #CTG GGC AAG GCG GTG CGA         488                                                                         Gly Gly Gly Pro Gly Arg Ala Gly Cys Cys - #Leu Gly Lys Ala Val Arg                        55     - #             60     - #             65                  - -  GGT GCC AAA GGT CAC CAC CAT CCC CAC CCG - #CCA GCC GCG GGC GCC GGC         536                                                                         Gly Ala Lys Gly His His His Pro His Pro - #Pro Ala Ala Gly Ala Gly                    70         - #         75         - #         80                      - -  GCG GCC GGG GGC GCC GAG GCG GAT CTG AAG - #GCG CTC ACG CAC TCG GTG         584                                                                         Ala Ala Gly Gly Ala Glu Ala Asp Leu Lys - #Ala Leu Thr His Ser Val                85             - #     90             - #     95                          - -  CTC AAG AAA CTG AAG GAG CGG CAG CTG GAG - #CTG CTG CTC CAG GCC GTG         632                                                                         Leu Lys Lys Leu Lys Glu Arg Gln Leu Glu - #Leu Leu Leu Gln Ala Val            100                 - #105                 - #110                 -         #115                                                                             - -  GAG TCC CGC GGC GGG ACG CGC ACC GCG TGC - #CTC CTG CTG CCC GGC        CGC     680                                                                      Glu Ser Arg Gly Gly Thr Arg Thr Ala Cys - #Leu Leu Leu Pro Gly Arg                           120 - #                125 - #                130             - -  CTG GAC TGC AGG CTG GGC CCG GGG GCG CCC - #GCC GGC GCG CAG CCT GCG         728                                                                         Leu Asp Cys Arg Leu Gly Pro Gly Ala Pro - #Ala Gly Ala Gln Pro Ala                        135     - #            140     - #            145                 - -  CAG CCG CCC TCG TCC TAC TCG CTC CCC CTC - #CTG CTG TGC AAA GTG TTC         776                                                                         Gln Pro Pro Ser Ser Tyr Ser Leu Pro Leu - #Leu Leu Cys Lys Val Phe                    150         - #        155         - #        160                     - -  AGG TGG CCG GAT CTC AGG CAT TCC TCG GAA - #GTC AAG AGG CTG TGT TGC         824                                                                         Arg Trp Pro Asp Leu Arg His Ser Ser Glu - #Val Lys Arg Leu Cys Cys                165             - #    170             - #    175                         - -  TGT GAA TCT TAC GGG AAG ATC AAC CCC GAG - #CTG GTG TGC TGC AAC CCC         872                                                                         Cys Glu Ser Tyr Gly Lys Ile Asn Pro Glu - #Leu Val Cys Cys Asn Pro            180                 - #185                 - #190                 -         #195                                                                             - -  CAT CAC CTT AGC CGA CTC TGC GAA CTA GAG - #TCT CCC CCC CCT CCT        TAC     920                                                                      His His Leu Ser Arg Leu Cys Glu Leu Glu - #Ser Pro Pro Pro Pro Tyr                           200 - #                205 - #                210             - -  TCC AGA TAC CCG ATG GAT TTT CTC AAA CCA - #ACT GCA GAC TGT CCA GAT         968                                                                         Ser Arg Tyr Pro Met Asp Phe Leu Lys Pro - #Thr Ala Asp Cys Pro Asp                        215     - #            220     - #            225                 - -  GCT GTG CCT TCC TCC GCT GAA ACA GGG GGA - #ACG AAT TAT CTG GCC CCT        1016                                                                         Ala Val Pro Ser Ser Ala Glu Thr Gly Gly - #Thr Asn Tyr Leu Ala Pro                    230         - #        235         - #        240                     - -  GGG GGG CTT TCA GAT TCC CAA CTT CTT CTG - #GAG CCT GGG GAT CGG TCA        1064                                                                         Gly Gly Leu Ser Asp Ser Gln Leu Leu Leu - #Glu Pro Gly Asp Arg Ser                245             - #    250             - #    255                         - -  CAC TGG TGC GTG GTG GCA TAC TGG GAG GAG - #AAG ACG AGA GTG GGG AGG        1112                                                                         His Trp Cys Val Val Ala Tyr Trp Glu Glu - #Lys Thr Arg Val Gly Arg            260                 - #265                 - #270                 -         #275                                                                             - -  CTC TAC TGT GTC CAG GAG CCC TCT CTG GAT - #ATC TTC TAT GAT CTA        CCT    1160                                                                      Leu Tyr Cys Val Gln Glu Pro Ser Leu Asp - #Ile Phe Tyr Asp Leu Pro                           280 - #                285 - #                290             - -  CAG GGG AAT GGC TTT TGC CTC GGA CAG CTC - #AAT TCG GAC AAC AAG AGT        1208                                                                         Gln Gly Asn Gly Phe Cys Leu Gly Gln Leu - #Asn Ser Asp Asn Lys Ser                        295     - #            300     - #            305                 - -  CAG CTG GTG CAG AAG GTG CGG AGC AAA ATC - #GGC TGC GGC ATC CAG CTG        1256                                                                         Gln Leu Val Gln Lys Val Arg Ser Lys Ile - #Gly Cys Gly Ile Gln Leu                    310         - #        315         - #        320                     - -  ACG CGG GAG GTG GAT GGT GTG TGG GTG TAC - #AAC CGC AGC AGT TAC CCC        1304                                                                         Thr Arg Glu Val Asp Gly Val Trp Val Tyr - #Asn Arg Ser Ser Tyr Pro                325             - #    330             - #    335                         - -  ATC TTC ATC AAG TCC GCC ACA CTG GAC AAC - #CCG GAC TCC AGG ACG CTG        1352                                                                         Ile Phe Ile Lys Ser Ala Thr Leu Asp Asn - #Pro Asp Ser Arg Thr Leu            340                 - #345                 - #350                 -         #355                                                                             - -  TTG GTA CAC AAG GTG TTC CCC GGT TTC TCC - #ATC AAG GCT TTC GAC        TAC    1400                                                                      Leu Val His Lys Val Phe Pro Gly Phe Ser - #Ile Lys Ala Phe Asp Tyr                           360 - #                365 - #                370             - -  GAG AAG GCG TAC AGC CTG CAG CGG CCC AAT - #GAC CAC GAG TTT ATG CAG        1448                                                                         Glu Lys Ala Tyr Ser Leu Gln Arg Pro Asn - #Asp His Glu Phe Met Gln                        375     - #            380     - #            385                 - -  CAG CCG TGG ACG GGC TTT ACC GTG CAG ATC - #AGC TTT GTG AAG GGC TGG        1496                                                                         Gln Pro Trp Thr Gly Phe Thr Val Gln Ile - #Ser Phe Val Lys Gly Trp                    390         - #        395         - #        400                     - -  GGT CAG TGC TAC ACC CGC CAG TTC ATC AGC - #AGC TGC CCG TGC TGG CTA        1544                                                                         Gly Gln Cys Tyr Thr Arg Gln Phe Ile Ser - #Ser Cys Pro Cys Trp Leu                405             - #    410             - #    415                         - -  GAG GTC ATC TTC AAC AGC CGG TAGCCGCGTG CGGAG - #GGGAC AGAGCGTGAG       CTGA  1599                                                                       Glu Val Ile Phe Asn Ser Arg                                                   420                 - #425                                                    - -  GCAGGCCACA CTTCAAACTA CTTTGCTGCT AATATTTTCC TCCTGAGTGC - #TTGCTTTT    CA  1659                                                                        - -  TGCAAACTCT TTGGTCGTTT TTTTTTTGTT TGTTGGTTGG TTTTCTTCTT - #CTCGTCCTC    G  1719                                                                         - -  TTTGTGTTCT GTTTTGTTTC GCTCTTTGAG AAATAGCTTA TGAAAAGAAT - #TGTTGGGGG    T  1779                                                                         - -  TTTTTTGGAA GAAGGGGCAG GTATGATCGG CAGGACACCC TGATAGGAAG - #AGGGGAAGC    A  1839                                                                         - -  GAAATCCAAG CACCACCAAA CACAGTGTAT GAAGGGGGGC GGTCATCATT - #TCACTTGTC    A  1899                                                                         - -  GGAGTGTGTG TGAGTGTGAG TGTGCGGCTG TGTGTGCACG CGTGTGCAGG - #AGCGGCAGA    T  1959                                                                         - -  GGGGAGACAA CGTGCTCTTT GTTTTGTGTC TCTTATGGAT GTCCCCAGCA - #GAGAGGTTT    G  2019                                                                         - -  CAGTCCCAAG CGGTGTCTCT CCTGCCCCTT GGACACGCTC AGTGGGGCAG - #AGGCAGTAC    C  2079                                                                         - -  TGGGCAAGCT GGCGGCTGGG GTCCCAGCAG CTGCCAGGAG CACGGCTCTG - #TCCCCAGCC    T  2139                                                                         - -  GGGAAAGCCC CTGCCCCTCC TCTCCCTCAT CAAGGACACG GGCCTGTCCA - #CAGGCTTCT    G  2199                                                                         - -  AGCAGCGAGC CTGCTAGTGG CCGAACCAGA ACCAATTATT TTCATCCTTG - #TCTTATTCC    C  2259                                                                         - -  TTCCTGCCAG CCCCTGCCAT TGTAGCGTCT TTCTTTTTTG GCCATCTGCT - #CCTGGATCT    C  2319                                                                         - -  CCTGAGATGG GCTTCCCAAG GGCTGCCGGG GCAGCCCCCT CACAGTATTG - #CTCACCCAG    T  2379                                                                         - -  GCCCTCTCCC CTCAGCCTCT CCCCTGCCTG CCCTGGTGAC ATCAGGTTTT - #TCCCGGACT    T  2439                                                                         - -  AGAAAACCAG CTCAGCACTG CCTGCTCCCA TCCTGTGTGT TAAGCTCTGC - #TATTAGGCC    A  2499                                                                         - -  GCAAGCGGGG ATGTCCCTGG GAGGGACATG CTTAGCAGTC CCCTTCCCTC - #CAAGAAGGA    T  2559                                                                         - -  TTGGTCCGTC ATAACCCAAG GTACCATCCT AGGCTGACAC CTAACTCTTC - #TTTCATTTC    T  2619                                                                         - -  TCTACAACTC ATACACTCGT ATGATACTTC GACACTGTTC TTAGCTCAAT - #GAGCATGTT    T  2679                                                                         - -  AGACTTTAAC ATAAGCTATT TTTCTAACTA CAAAGGTTTA AATGAACAAG - #AGAAGCATT    C  2739                                                                         - -  TCATTGGAAA TTTAGCATTG TAGTGCTTTG AGAGAGAAAG GACTCCTGAA - #AAAAAACCT    G  2799                                                                         - -  AGATTTATTA AAGAAAAAAA TGTATTTTAT GTTATATATA AATATATTAT - #TACTTGTAA    A  2859                                                                         - -  TATAAAGACG TTTTATAAGC ATCATTATTT ATGTATTGTG CAATGTGTAT - #AAACAAGAA    A  2919                                                                         - -  AATAAAGAAA AGATGCACTT TGCTTTAATA TAAATGCAAA TAACAAATGC - #CAAATTAAA    A  2979                                                                         - -  AAGATAAACA CAAGATTGGT GTTTTTTCCT ATGGGTGTTA TCACCTAGCT - #GAATGTTTT    T  3039                                                                         - -  CTAAAGGAGT TTATGTTCCA TTAAACGATT TTTAAAATGT ACACTTGAAA - #AAAAAAAAA    A  3099                                                                         - -  AAAA                - #                  - #                  - #               3103                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 426 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -  Met Phe Arg Thr Lys Arg Ser Ala Leu Val - #Arg Arg Leu Trp Arg Ser        1               5 - #                 10 - #                 15              - -  Arg Ala Pro Gly Gly Glu Asp Glu Glu Glu - #Gly Ala Gly Gly Gly Gly                   20     - #             25     - #             30                  - -  Gly Gly Gly Glu Leu Arg Gly Glu Gly Ala - #Thr Asp Ser Arg Ala His               35         - #         40         - #         45                      - -  Gly Ala Gly Gly Gly Gly Pro Gly Arg Ala - #Gly Cys Cys Leu Gly Lys           50             - #     55             - #     60                          - -  Ala Val Arg Gly Ala Lys Gly His His His - #Pro His Pro Pro Ala Ala       65                 - # 70                 - # 75                 - # 80       - -  Gly Ala Gly Ala Ala Gly Gly Ala Glu Ala - #Asp Leu Lys Ala Leu Thr                       85 - #                 90 - #                 95              - -  His Ser Val Leu Lys Lys Leu Lys Glu Arg - #Gln Leu Glu Leu Leu Leu                   100     - #            105     - #            110                 - -  Gln Ala Val Glu Ser Arg Gly Gly Thr Arg - #Thr Ala Cys Leu Leu Leu               115         - #        120         - #        125                     - -  Pro Gly Arg Leu Asp Cys Arg Leu Gly Pro - #Gly Ala Pro Ala Gly Ala           130             - #    135             - #    140                         - -  Gln Pro Ala Gln Pro Pro Ser Ser Tyr Ser - #Leu Pro Leu Leu Leu Cys       145                 - #150                 - #155                 -         #160                                                                             - -  Lys Val Phe Arg Trp Pro Asp Leu Arg His - #Ser Ser Glu Val Lys        Arg                                                                                              165 - #                170 - #                175            - -  Leu Cys Cys Cys Glu Ser Tyr Gly Lys Ile - #Asn Pro Glu Leu Val Cys                   180     - #            185     - #            190                 - -  Cys Asn Pro His His Leu Ser Arg Leu Cys - #Glu Leu Glu Ser Pro Pro               195         - #        200         - #        205                     - -  Pro Pro Tyr Ser Arg Tyr Pro Met Asp Phe - #Leu Lys Pro Thr Ala Asp           210             - #    215             - #    220                         - -  Cys Pro Asp Ala Val Pro Ser Ser Ala Glu - #Thr Gly Gly Thr Asn Tyr       225                 - #230                 - #235                 -         #240                                                                             - -  Leu Ala Pro Gly Gly Leu Ser Asp Ser Gln - #Leu Leu Leu Glu Pro        Gly                                                                                              245 - #                250 - #                255            - -  Asp Arg Ser His Trp Cys Val Val Ala Tyr - #Trp Glu Glu Lys Thr Arg                   260     - #            265     - #            270                 - -  Val Gly Arg Leu Tyr Cys Val Gln Glu Pro - #Ser Leu Asp Ile Phe Tyr               275         - #        280         - #        285                     - -  Asp Leu Pro Gln Gly Asn Gly Phe Cys Leu - #Gly Gln Leu Asn Ser Asp           290             - #    295             - #    300                         - -  Asn Lys Ser Gln Leu Val Gln Lys Val Arg - #Ser Lys Ile Gly Cys Gly       305                 - #310                 - #315                 -         #320                                                                             - -  Ile Gln Leu Thr Arg Glu Val Asp Gly Val - #Trp Val Tyr Asn Arg        Ser                                                                                              325 - #                330 - #                335            - -  Ser Tyr Pro Ile Phe Ile Lys Ser Ala Thr - #Leu Asp Asn Pro Asp Ser                   340     - #            345     - #            350                 - -  Arg Thr Leu Leu Val His Lys Val Phe Pro - #Gly Phe Ser Ile Lys Ala               355         - #        360         - #        365                     - -  Phe Asp Tyr Glu Lys Ala Tyr Ser Leu Gln - #Arg Pro Asn Asp His Glu           370             - #    375             - #    380                         - -  Phe Met Gln Gln Pro Trp Thr Gly Phe Thr - #Val Gln Ile Ser Phe Val       385                 - #390                 - #395                 -         #400                                                                             - -  Lys Gly Trp Gly Gln Cys Tyr Thr Arg Gln - #Phe Ile Ser Ser Cys        Pro                                                                                              405 - #                410 - #                415            - -  Cys Trp Leu Glu Val Ile Phe Asn Ser Arg                                              420     - #            425                                        - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1393 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Coding Se - #quence                                             (B) LOCATION: 90...938                                                        (D) OTHER INFORMATION:                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -  GGCACGAGGT TGCCCTGGCG GAGCAGAGAC AGGCCCTCGG GGTGGAGGTC - #TTTGGTTTC    A    60                                                                         - -  TAAGAGCCTG AGAGAGATTT TTCTAAGAT ATG TGT AAC ACA - #CCA ACG TAC TGT         113                                                                                          - #               Met Cys - #Asn Thr Pro Thr Tyr Cys                          - #                1  - #             5                      - -  GAC CTA GGA AAG GCT GCT AAG GAT GTC TTC - #AAC AAA GGA TAT GGC TTT         161                                                                         Asp Leu Gly Lys Ala Ala Lys Asp Val Phe - #Asn Lys Gly Tyr Gly Phe                10             - #     15             - #     20                          - -  GGC ATG GTC AAG ATA GAC CTG AAA ACC AAG - #TCT TGT AGT GGA GTG GAA         209                                                                         Gly Met Val Lys Ile Asp Leu Lys Thr Lys - #Ser Cys Ser Gly Val Glu            25                 - # 30                 - # 35                 - # 40       - -  TTT TCT ACT TCT GGT CAT GCT TAC ACT GAT - #ACA GGG AAA GCA TCA GGC         257                                                                         Phe Ser Thr Ser Gly His Ala Tyr Thr Asp - #Thr Gly Lys Ala Ser Gly                            45 - #                 50 - #                 55              - -  AAC CTA GAA ACC AAA TAT AAG GTC TGT AAC - #TAT GGA CTT ACC TTC ACC         305                                                                         Asn Leu Glu Thr Lys Tyr Lys Val Cys Asn - #Tyr Gly Leu Thr Phe Thr                        60     - #             65     - #             70                  - -  CAG AAA TGG AAC ACA GAC AAT ACT CTA GGG - #ACA GAA ATC TCT TGG GAG         353                                                                         Gln Lys Trp Asn Thr Asp Asn Thr Leu Gly - #Thr Glu Ile Ser Trp Glu                    75         - #         80         - #         85                      - -  AAT AAG TTG GCT GAA GGG TTG AAA CTG ACT - #CTT GAT ACC ATA TTT GTA         401                                                                         Asn Lys Leu Ala Glu Gly Leu Lys Leu Thr - #Leu Asp Thr Ile Phe Val                90             - #     95             - #     100                         - -  CCG AAC ACA GGA AAG AAG AGT GGG AAA TTG - #AAG GCC TCC TAT AAA CGG         449                                                                         Pro Asn Thr Gly Lys Lys Ser Gly Lys Leu - #Lys Ala Ser Tyr Lys Arg            105                 - #110                 - #115                 -         #120                                                                             - -  GAT TGT TTT AGT GTT GGC AGT AAT GTT GAT - #ATA GAT TTT TCT GGA        CCA     497                                                                      Asp Cys Phe Ser Val Gly Ser Asn Val Asp - #Ile Asp Phe Ser Gly Pro                           125 - #                130 - #                135             - -  ACC ATC TAT GGC TGG GCT GTG TTG GCC TTC - #GAA GGG TGG CTT GCT GGC         545                                                                         Thr Ile Tyr Gly Trp Ala Val Leu Ala Phe - #Glu Gly Trp Leu Ala Gly                        140     - #            145     - #            150                 - -  TAT CAG ATG AGT TTT GAC ACA GCC AAA TCC - #AAA CTG TCA CAG AAT AAT         593                                                                         Tyr Gln Met Ser Phe Asp Thr Ala Lys Ser - #Lys Leu Ser Gln Asn Asn                    155         - #        160         - #        165                     - -  TTC GCC CTG GGT TAC AAG GCT GCG GAC TTC - #CAG CTG CAC ACA CAT GTG         641                                                                         Phe Ala Leu Gly Tyr Lys Ala Ala Asp Phe - #Gln Leu His Thr His Val                170             - #    175             - #    180                         - -  AAC GAT GGC ACT GAA TTT GGA GGT TCT ATC - #TAC CAG AAG GTG AAT GAG         689                                                                         Asn Asp Gly Thr Glu Phe Gly Gly Ser Ile - #Tyr Gln Lys Val Asn Glu            185                 - #190                 - #195                 -         #200                                                                             - -  AAG ATT GAA ACA TCC ATA AAC CTT GCT TGG - #ACA GCT GGG AGT AAC        AAC     737                                                                      Lys Ile Glu Thr Ser Ile Asn Leu Ala Trp - #Thr Ala Gly Ser Asn Asn                           205 - #                210 - #                215             - -  ACC CGT TTT GGC ATT GCT GCT AAG TAC ATG - #CTG GAT TGT AGA ACT TCT         785                                                                         Thr Arg Phe Gly Ile Ala Ala Lys Tyr Met - #Leu Asp Cys Arg Thr Ser                        220     - #            225     - #            230                 - -  CTC TCT GCT AAA GTA AAT AAT GCC AGC CTG - #ATT GGA CTG GGT TAT ACT         833                                                                         Leu Ser Ala Lys Val Asn Asn Ala Ser Leu - #Ile Gly Leu Gly Tyr Thr                    235         - #        240         - #        245                     - -  CAG ACC CTT CGA CCA GGA GTC AAA TTG ACT - #TTA TCA GCT TTA ATC GAT         881                                                                         Gln Thr Leu Arg Pro Gly Val Lys Leu Thr - #Leu Ser Ala Leu Ile Asp                250             - #    255             - #    260                         - -  GGG AAG AAC TTC AGT GCA GGA GGT CAC AAG - #GTT GGC TTG GGA TTT GAA         929                                                                         Gly Lys Asn Phe Ser Ala Gly Gly His Lys - #Val Gly Leu Gly Phe Glu            265                 - #270                 - #275                 -         #280                                                                             - -  CTG GAA GCT TAATGTGGTT TGAGGAAAGC ATCAGATTTG TCCCT - #GGAAG           TGAAGAGAA   987                                                                  Leu Glu Ala                                                                   ATGAACCCAC TATGTTTTGG CCTTAAAATT CTTCTGTGAA ATTTCAAAAG - #TGTGAACTTT       1047                                                                             - -  TTATTCTTCC AAAGAATTGT AATCCTCCCC ACACTGAAGT CTAGGGGTTG - #CGAATCCC    TC  1107                                                                        - -  CTGAGGGAGA CGCTTGAAGG CATGCCTGGA AGTTGTCATG TTTGTGCCAC - #GTTTCAGTT    C  1167                                                                         - -  AGTTCTGAAG TGTTATTAAA TGTGTTCCTC AGCGACAGTG TAGCGTCATG - #TTAGAGGAG    A  1227                                                                         - -  CGATCTGACC CACCAGTTTG TACATCACGT CCTGCATGTC CCACACCATT - #TTTTCATGA    C  1287                                                                         - -  CTTGTAATAT ACTGGTCTCT GTGCTATAGT GGAATCTTTG GTTTTGCATC - #ATAGTAAAA    T  1347                                                                         - -  AAAATAAACC CATCACATTT GGAACATAAA AAAAAAAAAA AAAAAA  - #                   1393                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 283 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - -  Met Cys Asn Thr Pro Thr Tyr Cys Asp Leu - #Gly Lys Ala Ala Lys Asp        1               5 - #                 10 - #                 15              - -  Val Phe Asn Lys Gly Tyr Gly Phe Gly Met - #Val Lys Ile Asp Leu Lys                   20     - #             25     - #             30                  - -  Thr Lys Ser Cys Ser Gly Val Glu Phe Ser - #Thr Ser Gly His Ala Tyr               35         - #         40         - #         45                      - -  Thr Asp Thr Gly Lys Ala Ser Gly Asn Leu - #Glu Thr Lys Tyr Lys Val           50             - #     55             - #     60                          - -  Cys Asn Tyr Gly Leu Thr Phe Thr Gln Lys - #Trp Asn Thr Asp Asn Thr       65                 - # 70                 - # 75                 - # 80       - -  Leu Gly Thr Glu Ile Ser Trp Glu Asn Lys - #Leu Ala Glu Gly Leu Lys                       85 - #                 90 - #                 95              - -  Leu Thr Leu Asp Thr Ile Phe Val Pro Asn - #Thr Gly Lys Lys Ser Gly                   100     - #            105     - #            110                 - -  Lys Leu Lys Ala Ser Tyr Lys Arg Asp Cys - #Phe Ser Val Gly Ser Asn               115         - #        120         - #        125                     - -  Val Asp Ile Asp Phe Ser Gly Pro Thr Ile - #Tyr Gly Trp Ala Val Leu           130             - #    135             - #    140                         - -  Ala Phe Glu Gly Trp Leu Ala Gly Tyr Gln - #Met Ser Phe Asp Thr Ala       145                 - #150                 - #155                 -         #160                                                                             - -  Lys Ser Lys Leu Ser Gln Asn Asn Phe Ala - #Leu Gly Tyr Lys Ala        Ala                                                                                              165 - #                170 - #                175            - -  Asp Phe Gln Leu His Thr His Val Asn Asp - #Gly Thr Glu Phe Gly Gly                   180     - #            185     - #            190                 - -  Ser Ile Tyr Gln Lys Val Asn Glu Lys Ile - #Glu Thr Ser Ile Asn Leu               195         - #        200         - #        205                     - -  Ala Trp Thr Ala Gly Ser Asn Asn Thr Arg - #Phe Gly Ile Ala Ala Lys           210             - #    215             - #    220                         - -  Tyr Met Leu Asp Cys Arg Thr Ser Leu Ser - #Ala Lys Val Asn Asn Ala       225                 - #230                 - #235                 -         #240                                                                             - -  Ser Leu Ile Gly Leu Gly Tyr Thr Gln Thr - #Leu Arg Pro Gly Val        Lys                                                                                              245 - #                250 - #                255            - -  Leu Thr Leu Ser Ala Leu Ile Asp Gly Lys - #Asn Phe Ser Ala Gly Gly                   260     - #            265     - #            270                 - -  His Lys Val Gly Leu Gly Phe Glu Leu Glu - #Ala                                   275         - #        280                                            - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1036 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Coding Se - #quence                                             (B) LOCATION: 1...546                                                         (D) OTHER INFORMATION:                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - -  ACG AGC CTA GCC CTG GTG CTC AAC CTG CTG - #CAG ATC CAG AGG AAT GTC          48                                                                         Thr Ser Leu Ala Leu Val Leu Asn Leu Leu - #Gln Ile Gln Arg Asn Val             1               5 - #                 10 - #                 15              - -  ACT CTC TTC CCC GAG GAG GTG ATC GCC ACC - #ATC TTT TCC TCC GCC TGG          96                                                                         Thr Leu Phe Pro Glu Glu Val Ile Ala Thr - #Ile Phe Ser Ser Ala Trp                        20     - #             25     - #             30                  - -  TGG GTC CCT CCC TGC TGC GGG ACA GCA GCT - #GCT GTT GTT GGC CTA CTG         144                                                                         Trp Val Pro Pro Cys Cys Gly Thr Ala Ala - #Ala Val Val Gly Leu Leu                    35         - #         40         - #         45                      - -  TAC CCC TGT ATC GAC AGT CAC CTC GGA GAA - #CCC CAC AAA TTT AAG AGA         192                                                                         Tyr Pro Cys Ile Asp Ser His Leu Gly Glu - #Pro His Lys Phe Lys Arg                50             - #     55             - #     60                          - -  GAA TGG GCC AGT GTC ATG CGC TGC ATA GCA - #GTT TTT GTT GGC ATT AAC         240                                                                         Glu Trp Ala Ser Val Met Arg Cys Ile Ala - #Val Phe Val Gly Ile Asn            65                 - # 70                 - # 75                 - # 80       - -  CAC GCC AGT GCT AAA TTG GAT TTT GCC AAT - #AAT GTC CAG CTG TCC TTG         288                                                                         His Ala Ser Ala Lys Leu Asp Phe Ala Asn - #Asn Val Gln Leu Ser Leu                            85 - #                 90 - #                 95              - -  ACT TTA GCA GCC CTA TCT TTG GGC CTT TGG - #TGG ACA TTT GAT CGT TCC         336                                                                         Thr Leu Ala Ala Leu Ser Leu Gly Leu Trp - #Trp Thr Phe Asp Arg Ser                        100     - #            105     - #            110                 - -  AGA AGT GGC CTT GGG CTG GGG ATC ACC ATA - #GCT TTT CTA GCT ACG CTG         384                                                                         Arg Ser Gly Leu Gly Leu Gly Ile Thr Ile - #Ala Phe Leu Ala Thr Leu                    115         - #        120         - #        125                     - -  ATC ACG CAG TTT CTC GTG TAT AAT GGT GTC - #TAT CAG TAT ACA TCC CCA         432                                                                         Ile Thr Gln Phe Leu Val Tyr Asn Gly Val - #Tyr Gln Tyr Thr Ser Pro                130             - #    135             - #    140                         - -  GAT TTC CTC TAT ATT CGT TCT TGG CTC CCT - #TGT ATA TTT TTC TCA GGA         480                                                                         Asp Phe Leu Tyr Ile Arg Ser Trp Leu Pro - #Cys Ile Phe Phe Ser Gly            145                 - #150                 - #155                 -         #160                                                                             - -  GGC GTC ACG GTG GGG AAC ATA GGA CGA CAG - #TTA GCT ATG GGT GTT        CCT     528                                                                      Gly Val Thr Val Gly Asn Ile Gly Arg Gln - #Leu Ala Met Gly Val Pro                           165 - #                170 - #                175             - -  GAA AAG CCC CAT AGT GAT TGAGTCTTCA AAACCACCGA - #TTCTGAGAGC            AAGGAAGA   584                                                                   Glu Lys Pro His Ser Asp                                                                   180                                                               - -  TTTTGGAAGA AAATCTGACT GTGGATTATG ACAAAGATTA TCTTTTTTCT - #TAAGTAAT    CT   644                                                                        - -  ATTTAGATCG GGCTGACTGT ACAAATGACT CCTGGAAAAA ACTCTTCACC - #TAGTCTAGA    A   704                                                                         - -  TAGGGAGGTG GAGAATGATG ACTTACCCTG AAGTCTTCCC TTGACTGCCC - #GCACTGGCG    C   764                                                                         - -  CTGTCTGTGC CCTGGAGCAT TCTGCCCAGG CTACGTGGGT TCAGGCAGGT - #GGCAGCTTC    C   824                                                                         - -  CAAGTATTCG ATTTCATTCA TGTGATTAAA ACAAGTTGCC ATATTTCAAA - #AAAAAAAAA    A   884                                                                         - -  AAAAMCTCGA GACCAACCCG CAGTTTTGTG TCAGTGCCCA AAGGAGGTAG - #GTTGATGGT    G   944                                                                         - -  CTTAACAAAC ATGAAGTATG GTGTAATAGG AATAATATTT ATCCNAAAGA - #TTTTTAAAA    A  1004                                                                         - -  TAGGGCTGTG TTTAAAAAAA AAAAAAAAAA AA      - #                  - #            1036                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 182 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - -  Thr Ser Leu Ala Leu Val Leu Asn Leu Leu - #Gln Ile Gln Arg Asn Val        1               5 - #                 10 - #                 15              - -  Thr Leu Phe Pro Glu Glu Val Ile Ala Thr - #Ile Phe Ser Ser Ala Trp                   20     - #             25     - #             30                  - -  Trp Val Pro Pro Cys Cys Gly Thr Ala Ala - #Ala Val Val Gly Leu Leu               35         - #         40         - #         45                      - -  Tyr Pro Cys Ile Asp Ser His Leu Gly Glu - #Pro His Lys Phe Lys Arg           50             - #     55             - #     60                          - -  Glu Trp Ala Ser Val Met Arg Cys Ile Ala - #Val Phe Val Gly Ile Asn       65                 - # 70                 - # 75                 - # 80       - -  His Ala Ser Ala Lys Leu Asp Phe Ala Asn - #Asn Val Gln Leu Ser Leu                       85 - #                 90 - #                 95              - -  Thr Leu Ala Ala Leu Ser Leu Gly Leu Trp - #Trp Thr Phe Asp Arg Ser                   100     - #            105     - #            110                 - -  Arg Ser Gly Leu Gly Leu Gly Ile Thr Ile - #Ala Phe Leu Ala Thr Leu               115         - #        120         - #        125                     - -  Ile Thr Gln Phe Leu Val Tyr Asn Gly Val - #Tyr Gln Tyr Thr Ser Pro           130             - #    135             - #    140                         - -  Asp Phe Leu Tyr Ile Arg Ser Trp Leu Pro - #Cys Ile Phe Phe Ser Gly       145                 - #150                 - #155                 -         #160                                                                             - -  Gly Val Thr Val Gly Asn Ile Gly Arg Gln - #Leu Ala Met Gly Val        Pro                                                                                              165 - #                170 - #                175            - -  Glu Lys Pro His Ser Asp                                                              180                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1228 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Coding Se - #quence                                             (B) LOCATION: 1...468                                                         (D) OTHER INFORMATION:                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - -  ATG TGT CAC TCT CGC AGC TGC CAC CCG ACC - #ATG ACC ATC CTG CAG GCC          48                                                                         Met Cys His Ser Arg Ser Cys His Pro Thr - #Met Thr Ile Leu Gln Ala             1               5 - #                 10 - #                 15              - -  CCG ACC CCG GCC CCC TCC ACC ATC CCG GGA - #CCC CGG CGG GGC TCC GGT          96                                                                         Pro Thr Pro Ala Pro Ser Thr Ile Pro Gly - #Pro Arg Arg Gly Ser Gly                        20     - #             25     - #             30                  - -  CCT GAG ATC TTC ACC TTC GAC CCT CTC CCG - #GAG CCC GCA GCG GCC CCT         144                                                                         Pro Glu Ile Phe Thr Phe Asp Pro Leu Pro - #Glu Pro Ala Ala Ala Pro                    35         - #         40         - #         45                      - -  GCC GGG CGC CCC AGC GCC TCT CGC GGG CAC - #CGA AAG CGC AGC CGC AGG         192                                                                         Ala Gly Arg Pro Ser Ala Ser Arg Gly His - #Arg Lys Arg Ser Arg Arg                50             - #     55             - #     60                          - -  GTT CTC TAC CCT CGA GTG GTC CGG CGC CAG - #CTG CCA GTC GAG GAA CCG         240                                                                         Val Leu Tyr Pro Arg Val Val Arg Arg Gln - #Leu Pro Val Glu Glu Pro            65                 - # 70                 - # 75                 - # 80       - -  AAC CCA GCC AAA AGG CTT CTC TTT CTG CTG - #CTC ACC ATC GTC TTC TGC         288                                                                         Asn Pro Ala Lys Arg Leu Leu Phe Leu Leu - #Leu Thr Ile Val Phe Cys                            85 - #                 90 - #                 95              - -  CAG ATC CTG ATG GCT GAA GAG GGT GTG CCG - #GCG CCC CTG CCT CCA GAG         336                                                                         Gln Ile Leu Met Ala Glu Glu Gly Val Pro - #Ala Pro Leu Pro Pro Glu                        100     - #            105     - #            110                 - -  GAC GCC CCT AAC GCC GCA TCC CTG GCG CCC - #ACC CCT GTG TCC CCC GTC         384                                                                         Asp Ala Pro Asn Ala Ala Ser Leu Ala Pro - #Thr Pro Val Ser Pro Val                    115         - #        120         - #        125                     - -  CTC GAG CCC TTT AAT CTG ACT TCG GAG CCC - #TCG GAC TAC GCT CTG GAC         432                                                                         Leu Glu Pro Phe Asn Leu Thr Ser Glu Pro - #Ser Asp Tyr Ala Leu Asp                130             - #    135             - #    140                         - -  CTC AGC ACT TTC CTC CAG CAA CAC CCG GCC - #GCC TTC TAACTGTGAC          TCCCCG   484                                                                     Leu Ser Thr Phe Leu Gln Gln His Pro Ala - #Ala Phe                            145                 - #150                 - #155                             - -  CACTCCCCAA AAAGAATCCG AAAAACCACA AAGAAACACC AGGCGTACCT - #GGTGCGCG    AG   544                                                                        - -  AGCGTATCCC CAACTGGGAC TTCCGAGGCA ACTTGAACTC AGAACACTAC - #AGCGGAGAC    G   604                                                                         - -  CCACCCGGTG CTTGAGGCGG GACCGAGGCG CACAGAGACC GAGGCGCATA - #GAGACCGAG    G   664                                                                         - -  CACAGCCCAG CTGGGGCTAG GCCCGGTGGG AAGGAGAGCG TCGTTAATTT - #ATTTCTTAT    T   724                                                                         - -  GCTCCTAATT AATATTTATA TGTATTTATG TACGTCCTCC TAGGTGATGG - #AGATGTGTA    C   784                                                                         - -  GTAATATTTA TTTTAACTTA TGCAAGGGTG TGAGATGTTC CCTCTGCTGT - #AAATGCAGG    T   844                                                                         - -  CTCTTGGTAT TTATTGAGCT TTGTGGGACT GGTGGAAGCA GGACACCTGG - #AACTGCGGC    A   904                                                                         - -  AAGTAGGAGA AGAAATGGGG AGGACTCGGG TGGGGGAGGA CGTCCCGGCT - #GGGATGAAG    T   964                                                                         - -  CTGGTGGTGG GTCGTAAGTT TAGGAGGTGA CTGCATCCTC CAGCATCTCA - #ACTCCGTCT    G  1024                                                                         - -  TCTACTGTGT GAGACTTCGG CGGACCATTA GGAATGAGAT CCGTGAGATC - #CTTCCATCT    T  1084                                                                         - -  CTTGAAGTCG CCTTTAGGGT GGCTGCGAGG TAGAGGGTTG GGGGTTGGTG - #GGCTGTCAC    G  1144                                                                         - -  GAGCGACTGT CGAGATCGCC TAGTATGTTC TGTGAACACA AATAAAATTG - #ATTTACTGT    C  1204                                                                         - -  AAAAAAAAAA AAAAAAAACT CGAG         - #                  - #                  1228                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 156 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - -  Met Cys His Ser Arg Ser Cys His Pro Thr - #Met Thr Ile Leu Gln Ala        1               5 - #                 10 - #                 15              - -  Pro Thr Pro Ala Pro Ser Thr Ile Pro Gly - #Pro Arg Arg Gly Ser Gly                   20     - #             25     - #             30                  - -  Pro Glu Ile Phe Thr Phe Asp Pro Leu Pro - #Glu Pro Ala Ala Ala Pro               35         - #         40         - #         45                      - -  Ala Gly Arg Pro Ser Ala Ser Arg Gly His - #Arg Lys Arg Ser Arg Arg           50             - #     55             - #     60                          - -  Val Leu Tyr Pro Arg Val Val Arg Arg Gln - #Leu Pro Val Glu Glu Pro       65                 - # 70                 - # 75                 - # 80       - -  Asn Pro Ala Lys Arg Leu Leu Phe Leu Leu - #Leu Thr Ile Val Phe Cys                       85 - #                 90 - #                 95              - -  Gln Ile Leu Met Ala Glu Glu Gly Val Pro - #Ala Pro Leu Pro Pro Glu                   100     - #            105     - #            110                 - -  Asp Ala Pro Asn Ala Ala Ser Leu Ala Pro - #Thr Pro Val Ser Pro Val               115         - #        120         - #        125                     - -  Leu Glu Pro Phe Asn Leu Thr Ser Glu Pro - #Ser Asp Tyr Ala Leu Asp           130             - #    135             - #    140                         - -  Leu Ser Thr Phe Leu Gln Gln His Pro Ala - #Ala Phe                       145                 - #150                 - #155                             - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3084 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Coding Se - #quence                                             (B) LOCATION: 1032...1736                                                     (D) OTHER INFORMATION:                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - -  GAATTCGGCA CGAGGMCAGG AGCTCCTTTW CTGCGTCTCC CATCATGGGG - #CTTAGGGTT    G    60                                                                         - -  AGTCTTCAGG TTCTGGGGGC AGGAAGGACG GGCACTCAGG AGGCCCCCTC - #CCCATCCAC    A   120                                                                         - -  GCCCCTCTTT GGGAGGGGGG AAACTTGGCA ACCCGGGAGG CATGTGGATC - #TTTTCCTAA    G   180                                                                         - -  CAAGATGCTG AGCTGGAAAG ATGGGGGTGT AAGGTAATGT CCCAAACTGA - #AACTTTGCC    A   240                                                                         - -  GGCACTGGGA GAGGCTGTGA ACTCTTTTCT GGCTTTAGAA TTTAGGTCTA - #GATCCCAAA    A   300                                                                         - -  GGCTAAGTAC CCCCTGGGGG CTAACCAGAG GCATGCCTGG GCTGAGCTGA - #ACCTTCTGG    T   360                                                                         - -  GCACTGGCCC CTGGCTGACT GCTCTTCTGC AGGAAGTTGG AGGAGATTCC - #TGAAGTTGA    T   420                                                                         - -  TCCTCAGGCT GGATGTCCAA GGGGGTTGGA GTTTCTGATG TCTTTCTGTC - #TCCCTCTCT    T   480                                                                         - -  TTCTTTCTCT CCCTACCAGG TCCACTTCTT TCAGAGGGGC CTGCGGTGCT - #CTAAAAGTT    C   540                                                                         - -  TCCTGTTAAA GTTTAGAGCA AATTGGTTAT TATTTTAAAA TCAATAAAAC - #TTTTAAAAG    T   600                                                                         - -  ACTAAGACAA CTTCTAAGAG GGGAGTGGAC AGAGGGCCTG GTGGCAGCTC - #ACAGTTTCT    T   660                                                                         - -  TTCTGACCTT TGGTCTCACC CACCAAGTGT CCCACCTGAG TGCCCACCTT - #GCCCACCTG    A   720                                                                         - -  GGTAATGCCC TGGGGCTCCA CCAGTCCAGA TCCACAGGGC GCARCCATGT - #GGGAGTGGC    G   780                                                                         - -  GCTGATTGTT ACCCAGTAGT GTTGATAGCA CATTATTCAT AACAGCCAAA - #GAGAGGAAG    C   840                                                                         - -  AACCCAAATG TCCATTAGCT GATAAATGGA TAAATGAAAT ATGGTACGTC - #CGAAGAATG    G   900                                                                         - -  AATATCATTC ACCCATGAAA AAGAACGAAG TCCAGCACCA AAACGTGCTA - #CAACATGGA    T   960                                                                         - -  GAACTTCGAT GACTTTGTGC CACATGAAAG AAGAAGCCAG CCACAAAAGG - #CCATATATT    G  1020                                                                         - -  TATGAAATGA A ATG TCC AGA ATG GGC AAA CCC - #ATA GAG ACA CAA AAA        TCT   1070                                                                                    Met Ser Ar - #g Met Gly Lys Pro Ile Glu Thr Gln Lys Ser                       1   - #            5      - #            10                     - -  CCG CCA CCT CCC TAC TCT CGG CTG TCT CCT - #CGC GAC GAG TAC AAG CCA        1118                                                                         Pro Pro Pro Pro Tyr Ser Arg Leu Ser Pro - #Arg Asp Glu Tyr Lys Pro                15             - #     20             - #     25                          - -  CTG GAT CTG TCC GAT TCC ACA TTG TCT TAC - #ACT GAA ACG GAG GCT ACC        1166                                                                         Leu Asp Leu Ser Asp Ser Thr Leu Ser Tyr - #Thr Glu Thr Glu Ala Thr            30                 - # 35                 - # 40                 - # 45       - -  AAC TCC CTC ATC ACT GCT CCG GGT GAA TTC - #TCA GAC GCC AGC ATG TCT        1214                                                                         Asn Ser Leu Ile Thr Ala Pro Gly Glu Phe - #Ser Asp Ala Ser Met Ser                            50 - #                 55 - #                 60              - -  CCG GAC GCC ACC AAG CCG AGC CAC TGG TGC - #AGC GTG GCG TAC TGG GAG        1262                                                                         Pro Asp Ala Thr Lys Pro Ser His Trp Cys - #Ser Val Ala Tyr Trp Glu                        65     - #             70     - #             75                  - -  CAC CGG ACG CGC GTG GGC CGC CTC TAT GCG - #GTG TAC GAC CAG GCC GTC        1310                                                                         His Arg Thr Arg Val Gly Arg Leu Tyr Ala - #Val Tyr Asp Gln Ala Val                    80         - #         85         - #         90                      - -  AGC ATC TTC TAC GAC CTA CCT CAG GGC AGC - #GGC TTC TGC CTG GGC CAG        1358                                                                         Ser Ile Phe Tyr Asp Leu Pro Gln Gly Ser - #Gly Phe Cys Leu Gly Gln                95             - #     100            - #     105                         - -  CTC AAC CTG GAG CAG CGC AGC GAG TCG GTG - #CGG CGA ACG CGC AGC AAG        1406                                                                         Leu Asn Leu Glu Gln Arg Ser Glu Ser Val - #Arg Arg Thr Arg Ser Lys            110                 - #115                 - #120                 -         #125                                                                             - -  ATC GGC TTC GGC ATC CTG CTC AGC AAG GAG - #CCC GAC GGC GTG TGG        GCC    1454                                                                      Ile Gly Phe Gly Ile Leu Leu Ser Lys Glu - #Pro Asp Gly Val Trp Ala                           130 - #                135 - #                140             - -  TAC AAC CGC GGC GAG CAC CCC ATC TTC GTC - #AAC TCC CCG ACG CTG GAC        1502                                                                         Tyr Asn Arg Gly Glu His Pro Ile Phe Val - #Asn Ser Pro Thr Leu Asp                        145     - #            150     - #            155                 - -  GCG CCC GGC GGC CGC GCC CTG GTC GTG CGC - #AAG GTG CCC CCC GGC TAC        1550                                                                         Ala Pro Gly Gly Arg Ala Leu Val Val Arg - #Lys Val Pro Pro Gly Tyr                    160         - #        165         - #        170                     - -  TCC ATC AAG GTG TTC GAC TTC GAG CGC TCG - #GGC CTG CAG CAC GCG CCC        1598                                                                         Ser Ile Lys Val Phe Asp Phe Glu Arg Ser - #Gly Leu Gln His Ala Pro                175             - #    180             - #    185                         - -  GAG CCC GAC GCC GCC GAC GGC CCC TAC GAC - #CCC AAC AGC GTC CGC ATC        1646                                                                         Glu Pro Asp Ala Ala Asp Gly Pro Tyr Asp - #Pro Asn Ser Val Arg Ile            190                 - #195                 - #200                 -         #205                                                                             - -  AGC TTC GCC AAG GGC TGG GGG CCC TGC TAC - #TCC CGG CAG TTC ATC        ACC    1694                                                                      Ser Phe Ala Lys Gly Trp Gly Pro Cys Tyr - #Ser Arg Gln Phe Ile Thr                           210 - #                215 - #                220             - -  TCC TGC CCC TGC TGG CTG GAG ATC CTC CTC - #AAC AAC CCC AGA             TAGTGGCGG  1745                                                                  Ser Cys Pro Cys Trp Leu Glu Ile Leu Leu - #Asn Asn Pro Arg                                225     - #            230     - #            235                 - -  CCCCGGCGGG AGGGGCGGGT GGGAGGCCGC GGCCACCGCC ACCTGCCGGC - #CTCGAGAG    GG  1805                                                                        - -  GCCGATGCCC AGAGACACAG CCCCCACGGA CAAAACCCCC CAGATATCAT - #CTACCTAGA    T  1865                                                                         - -  TTAATATAAA GTTTTATATA TTATATGGAA ATATATATTA TACTTGTAAT - #TATGGAGTC    A  1925                                                                         - -  TTTTTACAAT GTAATTATTT ATGTATGGTG CAATGTGTGT ATATGGACAA - #AACAAGAAA    G  1985                                                                         - -  ACGCACTTTG GCTTATAATT CTTTCAATAC AGATATATTT TCTTTCTCTT - #CCTCCTTCC    T  2045                                                                         - -  CTTCCTTACT TTTTATATAT ATATATAAAG AAAATGATAC AGCAGAGCTA - #GGTGGAAAA    G  2105                                                                         - -  CCTGGGTTTG GTGTATGGTT TTTGAGATAT TAATGCCCAG ACAAAAAGCT - #AATACCAGT    C  2165                                                                         - -  ACTCGATAAT AAAGTATTCG CATTATAGTT TTTTTTAAAC TGTCTTCTTT - #TTACAAAGA    G  2225                                                                         - -  GGGCAGGTAG GGCTTCAGCG GATTTCTGAC CCATCATGTA CCTTGAAACT - #TGACCTCAG    T  2285                                                                         - -  TTTCAAGTTT TACTTTTATT GGATAAAGAC AGAACAAATT GAAAAGGGAG - #GAAAGTCAC    A  2345                                                                         - -  TTTACTCTTA AGTAAACCAG AGAAAGTTCT GTTGTTCCTT CCTGCCCATG - #GCTATGGGG    T  2405                                                                         - -  GTCCAGTGGA TAGGGATGGC GGTGGGGAAA AGGAGAATAC ACTGGCCATT - #TATCCTGGA    C  2465                                                                         - -  AAGCTCTTCC AGTCTGATGG AGGAGGTTCA TGCCCTAGCC TAGAAAGGCC - #CAGGTCCAT    G  2525                                                                         - -  ACCCCCATCT TTGAGTTATG AGCAAGCTAA AAGAAGACAC TATTTCTCAC - #CATTTTGTG    G  2585                                                                         - -  AAATGGCCTG GGGAACAAAG ACTGAAATGG GCCTTGAGCC CACCTGCTAC - #CTTGCAGAG    A  2645                                                                         - -  ACCATCTCGA GCCCCGTAGA TCTTTTTAGG ACCTCCACAG GSTATTTCCC - #ACCCCCCAG    C  2705                                                                         - -  CAAAAATAGC TCAGAATCTG CCCATCCAGG GCTTGTATTA ATGATTTATG - #TAAAGGCAG    A  2765                                                                         - -  TGGTTTATTT CTACTTTGTA AAAGGGAAAA GTTGAGGTTC TGGAAGGATA - #AATGATTTG    C  2825                                                                         - -  TCATGAGACA AAATCAAGGT TAGAAGTTAC ATGGAATTGT AGGACCAGAG - #CCATATCAT    T  2885                                                                         - -  AGATCAGCTT TCTGAAGAAT ATTCTCCAMA AAAGAAAGTC TCCTTGGCCA - #GATAACTAA    G  2945                                                                         - -  AGGAATGTTT CATTGTATAT CTTTTTTCTT GGAGATTTAT ATTAACATAT - #TAAGTGCTC    T  3005                                                                         - -  GAGAAGTCCT GTGTATTATC TCTTGCTGCA TAATAAATTA TCCCCAMACT - #TAAAAAAAA    A  3065                                                                         - -  AAAAAAAAAA AAACTCGAG            - #                  - #                     308 - #4                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 235 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - -  Met Ser Arg Met Gly Lys Pro Ile Glu Thr - #Gln Lys Ser Pro Pro Pro        1               5 - #                 10 - #                 15              - -  Pro Tyr Ser Arg Leu Ser Pro Arg Asp Glu - #Tyr Lys Pro Leu Asp Leu                   20     - #             25     - #             30                  - -  Ser Asp Ser Thr Leu Ser Tyr Thr Glu Thr - #Glu Ala Thr Asn Ser Leu               35         - #         40         - #         45                      - -  Ile Thr Ala Pro Gly Glu Phe Ser Asp Ala - #Ser Met Ser Pro Asp Ala           50             - #     55             - #     60                          - -  Thr Lys Pro Ser His Trp Cys Ser Val Ala - #Tyr Trp Glu His Arg Thr       65                 - # 70                 - # 75                 - # 80       - -  Arg Val Gly Arg Leu Tyr Ala Val Tyr Asp - #Gln Ala Val Ser Ile Phe                       85 - #                 90 - #                 95              - -  Tyr Asp Leu Pro Gln Gly Ser Gly Phe Cys - #Leu Gly Gln Leu Asn Leu                   100     - #            105     - #            110                 - -  Glu Gln Arg Ser Glu Ser Val Arg Arg Thr - #Arg Ser Lys Ile Gly Phe               115         - #        120         - #        125                     - -  Gly Ile Leu Leu Ser Lys Glu Pro Asp Gly - #Val Trp Ala Tyr Asn Arg           130             - #    135             - #    140                         - -  Gly Glu His Pro Ile Phe Val Asn Ser Pro - #Thr Leu Asp Ala Pro Gly       145                 - #150                 - #155                 -         #160                                                                             - -  Gly Arg Ala Leu Val Val Arg Lys Val Pro - #Pro Gly Tyr Ser Ile        Lys                                                                                              165 - #                170 - #                175            - -  Val Phe Asp Phe Glu Arg Ser Gly Leu Gln - #His Ala Pro Glu Pro Asp                   180     - #            185     - #            190                 - -  Ala Ala Asp Gly Pro Tyr Asp Pro Asn Ser - #Val Arg Ile Ser Phe Ala               195         - #        200         - #        205                     - -  Lys Gly Trp Gly Pro Cys Tyr Ser Arg Gln - #Phe Ile Thr Ser Cys Pro           210             - #    215             - #    220                         - -  Cys Trp Leu Glu Ile Leu Leu Asn Asn Pro - #Arg                           225                 - #230                 - #235                             - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - -  TTTTTTTTTT TNC             - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - -  GTGAGGCGTC               - #                  - #                      - #        10                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - -  TGGACCGGTG               - #                  - #                      - #        10                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - -  TTTTTTTTTT TNA             - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - -  AGACGTCCAC               - #                  - #                      - #        10                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - -  ACTTCGCCAC               - #                  - #                      - #        10                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - -  TCGGACGTGA               - #                  - #                      - #        10                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - -  CATTTCATTT CATACAA            - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - -  CATTTCATTT CATACAATAT ATG          - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - -  CATTTCATTT CATACAATAT ATGGCCTTT        - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - -  CATTTCATTT CATACAATAT ATGGCCTTTT GTGGC      - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - -  GGACATTTCA TTTCATACAA TATATGGCCT TTTGT      - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - -  TTCATTTCAT ACAATATATG GCCTTTTGT        - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - -  TCATACAATA TATGGCCTTT TGT          - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - -  AATATATGGC CTTTTGT            - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - -  CATGCGGGGC GAGGAGG            - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - -  CATGCGGGGC GAGGAGGCGA GGA          - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - -  CATGCGGGGC GAGGAGGCGA GGAGAAAAG        - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - -  CATGCGGGGC GAGGAGGCGA GGAGAAAAGT CGTTT      - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - -  GAACATGCGG GGCGAGGAGG CGAGGAGAAA AGTCG      - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - -  GCGGGGCGAG GAGGCGAGGA GAAAAGTCG        - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - -  CGAGGAGGCG AGGAGAAAAG TCG          - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - -  GGCGAGGAGA AAAGTCG            - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - -  CAAAGCNGNN NNNNCNGAGN AGUC         - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - -  GGUGGAGCCC CAGGGCAUUA CCUCAAAGCN GNNNNNNCNG AGNAGUCGUG - #GGCAAGGUG    G    60                                                                         - -  GCACUCAGGU GGG             - #                  - #                      - #      73                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - -  GUGUCUCUAU GGGUUUGCCC AAAGCNGNNN NNNCNGAGNA GUCUCUGGAC - #AUUUCAUUU    C    60                                                                         - -  AUAC                - #                  - #                  - #                 64                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - -  GGCCCUCUCG CCGUCGGGCU CCUUGCUGAG CAAAGCNGNN NNNNCNGAGN - #AGUCGAUGC    C    60                                                                         - -  GAAGCCGAUC UUGCUGCGCG           - #                  - #                      - # 80                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - -  CGUUUGCCUG CUAAGGAGCG AACAAAGCNG NNNNNNCNGA GNAGUCGAUG - #UUUCUUUGU    G    60                                                                         - -  AGUCGGGCGC CG             - #                  - #                      - #       72                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 84 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - -  CGCCGGACGA GCGCAGAUCG UUUGGUCCUG AACAAAGCNG NNNNNNCNGA - #GNAGUCCGG    G    60                                                                         - -  GCGAGGAGGC GAGGAGAAAA GUCG         - #                  - #                    84                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 88 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - -  GGAGUAAGGA GGGGGGGGAG ACUCUAGUUC GCAAAGCNGN NNNNNCNGAG - #NAGUCAGUC    G    60                                                                         - -  GCUAAGGUGA UGGGGGUUGC AGCACACC        - #                  - #                 88                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - -  Tyr Thr Asp Thr Gly Lys Ala Ser Gly Asn - #Leu Glu Thr Lys Tyr Lys        1               5 - #                 10 - #                 15              - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - -  Thr Gly Lys Lys Ser Gly Lys Leu Lys Ala - #Ser Tyr Lys Arg Asp            1               5 - #                 10 - #                 15            __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide comprising anucleotide sequence: (a) encoding a polypeptide having the fchd540 aminoacid sequence set forth in SEQ ID NO:4; or (b) encoding a polypeptideencoded by the fchd540 cDNA contained in plasmid pFCHD540, as depositedwith the ATCC as ATCC Accession No. 69984; or (c) which is thecomplement of (a) or (b).
 2. An isolated polynucleotide comprising thefchd540 nucleotide sequence: (a) set forth in SEQ ID NO:3; or (b) of thefchd540 cDNA contained in plasmid pFCHD540, as deposited with the ATCCas ATCC Accession No. 69984; or (c) which is the complement of (a). 3.An isolated polynucleotide comprising the nucleotide sequence: (a) ofthe fchd540 polypeptide coding sequence, as set forth from nucleotideresidue number 288 to 1565 of SEQ ID NO:3; or (b) of the fchd540polypeptide coding sequence of the fchd540 cDNA contained in plasmidpFCHD540, as deposited with the ATCC as ATCC Accession No. 69984; or (c)which is the complement of (a) or (b).
 4. An isolated polynucleotide ofclaim 1, 2 or 3 which is DNA.
 5. The isolated polynucleotide of claim 4which is cDNA.
 6. The isolated polynucleotide of claim 1, 2, or 3 whichis RNA.
 7. The isolated polynucleotide of claim 1, 2, or 3 which furthercomprises a label.
 8. A polynucleotide vector containing thepolynucleotide of claim 1, 2, or
 3. 9. A polynucleotide expressionvector containing the polynucleotide of claim 1, 2, or 3 in operativeassociation with a nucleotide regulatory element which controlsexpression of the polynucleotide in a host cell.
 10. A geneticallyengineered host cell containing the polynucleotide of claim 1, 2, or 3in association with nucleotide sequences exogenous to thepolynucleotide.
 11. A genetically engineered host cell containing thepolynucleotide of claim 1, 2, or 3 in operative association with anucleotide regulatory element exogenous to the polynucleotide, whereinthe regulatory element controls expression of the polynucleotide in thehost cell.
 12. The genetically engineered host cell of claim 11 which isprokaryotic.
 13. The genetically engineered host cell of claim 11 whichis eukaryotic.
 14. A method of producing a polypeptide fchd540 geneproduct, comprising the steps of:(a) growing the genetically engineeredhost cell of claim 12 in a cultures; and (b) collecting the polypeptidegene product from the culture.
 15. A method of producing a polypeptidefchd540 gene product, comprising the steps of:(a) growing thegenetically engineered host cell of claim 13 in a culture; and (b)collecting the polypeptide gene product from the culture.